Non-liposomal systems for nucleic acid delivery

ABSTRACT

The present invention provides novel, stable lipid particles having a non-lamellar structure and comprising one or more active agents or therapeutic agents, methods of making such lipid particles, and methods of delivering and/or administering such lipid particles. More particularly, the present invention provides stable nucleic acid-lipid particles (SNALP) that have a non-lamellar structure and that comprise a nucleic acid (such as one or more interfering RNA), methods of making the SNALP, and methods of delivering and/or administering the SNALP.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.14/642,452, filed Mar. 9, 2015, which is a continuation of U.S.application Ser. No. 13/807,288, filed Apr. 18, 2013, and which issuedon Apr. 14, 2015, as U.S. Pat. No. 9,006,417 B2, which application is aNational Phase application under 35 U.S.C. §371 of PCT/CA2011/000778,filed Jun. 30, 2011, which application claims the benefit of U.S.Provisional Application No. 61/360,480, filed Jun. 30, 2010, thedisclosures of which are incorporated herein by reference for allpurposes.

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file -100-1.TXT, created on May 15,2013, 4,096 bytes, machine format IBM-PC, MS-Windows operating system,is hereby incorporated by reference in its entirety for all purposes.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is an evolutionarily conserved process in whichrecognition of double-stranded RNA (dsRNA) ultimately leads toposttranscriptional suppression of gene expression. This suppression ismediated by short dsRNA, also called small interfering RNA (siRNA),which induces specific degradation of mRNA through complementary basepairing. In several model systems, this natural response has beendeveloped into a powerful tool for the investigation of gene function(see, e.g., Elbashir et al., Genes Dev., 15:188-200 (2001); Hammond etal., Nat. Rev. Genet., 2:110-119 (2001)). More recently, it wasdiscovered that introducing synthetic 21-nucleotide dsRNA duplexes intomammalian cells could efficiently silence gene expression.

Although the precise mechanism is still unclear, RNAi provides apotential new approach to downregulate or silence the transcription andtranslation of a gene of interest. For example, it is desirable tomodulate (e.g., reduce) the expression of certain genes for thetreatment of neoplastic disorders such as cancer. It is also desirableto silence the expression of genes associated with liver diseases anddisorders such as hepatitis. It is further desirable to reduce theexpression of certain genes for the treatment of atherosclerosis and itsmanifestations, e.g., hypercholesterolemia, myocardial infarction, andthrombosis.

A safe and effective nucleic acid delivery system is required for RNAito be therapeutically useful. Viral vectors are relatively efficientgene delivery systems, but suffer from a variety of limitations, such asthe potential for reversion to the wild-type as well as immune responseconcerns. As a result, nonviral gene delivery systems are receivingincreasing attention (Worgall et al., Human Gene Therapy, 8:37 (1997);Peeters et al., Human Gene Therapy, 7:1693 (1996); Yei et al., GeneTherapy, 1:192 (1994); Hope et al., Molecular Membrane Biology, 15:1(1998)). Furthermore, viral systems are rapidly cleared from thecirculation, limiting transfection to “first-pass” organs such as thelungs, liver, and spleen. In addition, these systems induce immuneresponses that compromise delivery with subsequent injections.

Plasmid DNA-cationic liposome complexes are currently the most commonlyemployed nonviral gene delivery vehicles (Felgner, Scientific American,276:102 (1997); Chonn et al., Current Opinion in Biotechnology, 6:698(1995)). For instance, cationic liposome complexes made of anamphipathic compound, a neutral lipid, and a detergent for transfectinginsect cells are disclosed in U.S. Pat. No. 6,458,382. Cationic liposomecomplexes are also disclosed in U.S. Patent Publication No. 20030073640.

Cationic liposome complexes are large, poorly defined systems that arenot suited for systemic applications and can elicit considerable toxicside effects (Harrison et al., Biotechniques, 19:816 (1995); Li et al.,The Gene, 4:891 (1997); Tam et al, Gene Ther 7:1867 (2000)). As large,positively charged aggregates, lipoplexes are rapidly cleared whenadministered in vivo, with highest expression levels observed infirst-pass organs, particularly the lungs (Huang et al., NatureBiotechnology, 15:620 (1997); Templeton et al., Nature Biotechnology,15:647 (1997); Hofland et al., Pharmaceutical Research, 14:742 (1997)).

Other liposomal delivery systems include, for example, the use ofreverse micelles, anionic liposomes, and polymer liposomes. Reversemicelles are disclosed in U.S. Pat. No. 6,429,200. Anionic liposomes aredisclosed in U.S. Patent Publication No. 20030026831. Polymer liposomesthat incorporate dextrin or glycerol-phosphocholine polymers aredisclosed in U.S. Patent Publication Nos. 20020081736 and 20030082103,respectively.

A gene delivery system containing an encapsulated nucleic acid forsystemic delivery should be small (i.e., less than about 100 nmdiameter) and should remain intact in the circulation for an extendedperiod of time in order to achieve delivery to affected tissues. Thisrequires a highly stable, serum-resistant nucleic acid-containingparticle that does not interact with cells and other components of thevascular compartment. The particle should also readily interact withtarget cells at a disease site in order to facilitate intracellulardelivery of a desired nucleic acid.

Recent work has shown that nucleic acids can be encapsulated in small(e.g., about 70 nm diameter) “stabilized plasmid-lipid particles” (SPLP)that consist of a single plasmid encapsulated within a bilayer lipidvesicle (Wheeler et al., Gene Therapy, 6:271 (1999)). These SPLPstypically contain the “fusogenic” lipid dioleoylphosphatidylethanolamine(DOPE), low levels of cationic lipid, and are stabilized in aqueousmedia by the presence of a poly(ethylene glycol) (PEG) coating. SPLPshave systemic application as they exhibit extended circulation lifetimesfollowing intravenous (i.v.) injection, accumulate preferentially atdistal tumor sites due to the enhanced vascular permeability in suchregions, and can mediate transgene expression at these tumor sites. Thelevels of transgene expression observed at the tumor site following i.v.injection of SPLPs containing the luciferase marker gene are superior tothe levels that can be achieved employing plasmid DNA-cationic liposomecomplexes (lipoplexes) or naked DNA.

Thus, there remains a strong need in the art for novel and moreefficient methods and compositions for introducing nucleic acids such assiRNA into cells. In addition, there is a need in the art for methods ofdownregulating the expression of genes of interest to treat or preventdiseases and disorders such as cancer and atherosclerosis. The presentinvention addresses these and other needs.

BRIEF SUMMARY OF THE INVENTION

The present invention is based, in part, upon the surprising discoverythat by controlling the lipid composition of a SNALP formulation as wellas the formation process used to prepare the SNALP formulation, a novelnon-lamellar lipid nanoparticle (i.e., SNALP) can be produced. Moreparticularly, it has surprisingly been found that lipid particles thatcomprise from about 50 mol % to about 85 mol % of a cationic lipid, fromabout 13 mol % to about 49.5 mol % of a non-cationic lipid, and fromabout 0.5 mol % to about 10 mol % of a lipid conjugate, and that aremade using the Direct Dilution Method as described herein have a novelnon-lamellar (i.e., non-bilayer) morphology and enhanced silencingability when used to deliver an interfering nucleic acid, such as ansiRNA molecule. As such, the present invention provides a compositioncomprising a plurality of nucleic acid-lipid particles, wherein eachparticle in the plurality of particles comprises: (a) a nucleic acid;(b) a cationic lipid comprising from about 50 mol % to about 85 mol % ofthe total lipid present in the particle; (c) a non-cationic lipidcomprising from about 13 mol % to about 49.5 mol % of the total lipidpresent in the particle; and (d) a conjugated lipid that inhibitsaggregation of particles comprising from about 0.5 mol % to about 10 mol% of the total lipid present in the particle, wherein at least about 95%of the particles in the plurality of particles have a non-lamellarmorphology. In preferred embodiments, greater than 95%, preferably,greater than 96%, preferably, greater than 97%, preferably, greater than98% and, preferably, greater than 99% of the particles have anon-lamellar morphology, i.e., a non-bilayer structure.

In certain embodiments, the active agent or therapeutic agent is fullyencapsulated within the lipid portion of the lipid particles such thatthe active agent or therapeutic agent in the lipid particle is resistantin aqueous solution to enzymatic degradation, e.g., by a nuclease orprotease. In certain other embodiments, the lipid particles aresubstantially non-toxic to mammals such as humans.

In some embodiments, the active agent or therapeutic agent comprises anucleic acid. In certain instances, the nucleic acid comprises aninterfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA, ormixtures thereof. In certain other instances, the nucleic acid comprisessingle-stranded or double-stranded DNA, RNA, or a DNA/RNA hybrid suchas, e.g., an antisense oligonucleotide, a ribozyme, a plasmid, animmunostimulatory oligonucleotide, or mixtures thereof.

In preferred embodiments, the active agent or therapeutic agentcomprises an siRNA. In one embodiment, the siRNA molecule comprises adouble-stranded region of about 15 to about 60 nucleotides in length(e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides inlength, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength). The siRNA molecules of the invention are capable of silencingthe expression of a target sequence in vitro and/or in vivo.

In some embodiments, the siRNA molecule comprises at least one modifiednucleotide. In certain preferred embodiments, the siRNA moleculecomprises one, two, three, four, five, six, seven, eight, nine, ten, ormore modified nucleotides in the double-stranded region. In certaininstances, the siRNA comprises from about 1% to about 100% (e.g., about1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in thedouble-stranded region. In preferred embodiments, less than about 25%(e.g., less than about 25%, 20%, 15%, 10%, or 5%) or from about 1% toabout 25% (e.g., from about 1%-25%, 5%-25%, 10%-25%, 15%-25%, 20%-25%,or 10%-20%) of the nucleotides in the double-stranded region comprisemodified nucleotides.

In other embodiments, the siRNA molecule comprises modified nucleotidesincluding, but not limited to, 2′-O-methyl (2′OMe) nucleotides,2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, and mixtures thereof. In preferred embodiments, the siRNAcomprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosinenucleotides, and mixtures thereof. In certain instances, the siRNA doesnot comprise 2′OMe-cytosine nucleotides. In other embodiments, the siRNAcomprises a hairpin loop structure.

The siRNA may comprise modified nucleotides in one strand (i.e., senseor antisense) or both strands of the double-stranded region of the siRNAmolecule. Preferably, uridine and/or guanosine nucleotides are modifiedat selective positions in the double-stranded region of the siRNAduplex. With regard to uridine nucleotide modifications, at least one,two, three, four, five, six, or more of the uridine nucleotides in thesense and/or antisense strand can be a modified uridine nucleotide suchas a 2′OMe-uridine nucleotide. In some embodiments, every uridinenucleotide in the sense and/or antisense strand is a 2′OMe-uridinenucleotide. With regard to guanosine nucleotide modifications, at leastone, two, three, four, five, six, or more of the guanosine nucleotidesin the sense and/or antisense strand can be a modified guanosinenucleotide such as a 2′OMe-guanosine nucleotide. In some embodiments,every guanosine nucleotide in the sense and/or antisense strand is a2′OMe-guanosine nucleotide.

In the lipid particles of the invention (e.g., SNALP comprising aninterfering RNA such as siRNA), the cationic lipid may comprise, e.g.,one or more of the following: the cationic lipids of Formula I asdisclosed herein, including, for example, MC3, LenMC3, CP-LenMC3,γ-LenMC3, CP-γ-LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide,Pan-MC3, Pan-MC4 and Pan MC5, 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane(DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)[1,3]-dioxolane(DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane(DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane(DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane(DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane(DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), ormixtures thereof. In certain preferred embodiments, the cationic lipidis DLinDMA, DLin-K-C2-DMA (“XTC2”), MC3, LenMC3, CP-LenMC3, γ-LenMC3,CP-γ-LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3,Pan-MC4, Pan MC5, or mixtures thereof.

In the lipid particles of the invention (e.g., SNALP comprising aninterfering RNA such as siRNA), the non-cationic lipid may comprise,e.g., one or more anionic lipids and/or neutral lipids. In preferredembodiments, the non-cationic lipid comprises one of the followingneutral lipid components: (1) cholesterol or a derivative thereof; (2) aphospholipid; or (3) a mixture of a phospholipid and cholesterol or aderivative thereof.

Examples of cholesterol derivatives include, but are not limited to,cholestanol, cholestanone, cholestenone, coprostanol,cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether,and mixtures thereof. The synthesis of cholesteryl-2′-hydroxyethyl etheris described herein.

The phospholipid may be a neutral lipid including, but not limited to,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine(EPC), and mixtures thereof. In certain preferred embodiments, thephospholipid is DPPC, DSPC, or mixtures thereof.

In the lipid particles of the invention (e.g., SNALP comprising aninterfering RNA such as siRNA), the conjugated lipid that inhibitsaggregation of particles may comprise, e.g., one or more of thefollowing: a polyethyleneglycol (PEG)-lipid conjugate, a polyamide(ATTA)-lipid conjugate, a cationic-polymer-lipid conjugates (CPLs), ormixtures thereof. In one preferred embodiment, the nucleic acid-lipidparticles comprise either a PEG-lipid conjugate or an ATTA-lipidconjugate. In certain embodiments, the PEG-lipid conjugate or ATTA-lipidconjugate is used together with a CPL. The conjugated lipid thatinhibits aggregation of particles may comprise a PEG-lipid including,e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAAconjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl(C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18),or mixtures thereof.

In one specific embodiment, the composition of the present inventioncomprises: a plurality of nucleic acid-lipid particles, wherein eachparticle in the plurality of particles comprises: (a) one or moreunmodified and/or modified interfering RNA (e.g., siRNA, aiRNA, miRNA)that silence target gene expression; (b) a cationic lipid comprisingfrom about 56.5 mol % to about 66.5 mol % of the total lipid present inthe particle; (c) a non-cationic lipid comprising from about 31.5 mol %to about 42.5 mol % of the total lipid present in the particle; and (d)a conjugated lipid that inhibits aggregation of particles comprisingfrom about 1 mol % to about 2 mol % of the total lipid present in theparticle, wherein at least about 95% of the particles in the pluralityof particles have a non-lamellar morphology. This specific embodiment ofSNALP is generally referred to herein as the “1:62” formulation. In apreferred embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA(“XTC2”), the non-cationic lipid is cholesterol, and the conjugatedlipid is a PEG-DAA conjugate. Although these are preferred embodimentsof the 1:62 formulation, those of skill in the art will appreciate thatother cationic lipids, non-cationic lipids (including other cholesterolderivatives), and conjugated lipids can be used in the 1:62 formulationas described herein.

In another specific embodiment, the composition of the present inventioncomprises: a plurality of nucleic acid-lipid particles, wherein eachparticle in the plurality of particles comprises: (a) one or moreunmodified and/or modified interfering RNA (e.g., siRNA, aiRNA, miRNA)that silence target gene expression; (b) a cationic lipid comprisingfrom about 52 mol % to about 62 mol % of the total lipid present in theparticle; (c) a non-cationic lipid comprising from about 36 mol % toabout 47 mol % of the total lipid present in the particle; and (d) aconjugated lipid that inhibits aggregation of particles comprising fromabout 1 mol % to about 2 mol % of the total lipid present in theparticle, wherein at least about 95% of the particles in the pluralityof particles have a non-lamellar morphology. This specific embodiment ofSNALP is generally referred to herein as the “1:57” formulation. In onepreferred embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA(“XTC2”), the non-cationic lipid is a mixture of a phospholipid (such asDPPC) and cholesterol, wherein the phospholipid comprises from about 5mol % to about 9 mol % of the total lipid present in the particle (e.g.,about 7.1 mol %) and the cholesterol (or cholesterol derivative)comprises from about 32 mol % to about 37 mol % of the total lipidpresent in the particle (e.g., about 34.3 mol %), and the PEG-lipid is aPEG-DAA (e.g., PEG-cDMA). In another preferred embodiment, the cationiclipid is DLinDMA or DLin-K-C2-DMA (“XTC2”), the non-cationic lipid is amixture of a phospholipid (such as DPPC) and cholesterol, wherein thephospholipid comprises from about 15 mol % to about 25 mol % of thetotal lipid present in the particle (e.g., about 20 mol %) and thecholesterol (or cholesterol derivative) comprises from about 15 mol % toabout 25 mol % of the total lipid present in the particle (e.g., about20 mol %), and the PEG-lipid is a PEG-DAA (e.g., PEG-cDMA). Althoughthese are preferred embodiments of the 1:57 formulation, those of skillin the art will appreciate that other cationic lipids, non-cationiclipids (including other phospholipids and other cholesterolderivatives), and conjugated lipids can be used in the 1:57 formulationas described herein.

In yet another specific embodiment, the composition of the presentinvention comprises: a plurality of nucleic acid-lipid particles,wherein each particle in the plurality of particles comprises: (a) anucleic acid (e.g., an interfering RNA); (b) a cationic lipid comprisingfrom about 50 mol % to about 60 mol % of the total lipid present in theparticle; (c) a mixture of a phospholipid and cholesterol or aderivative thereof comprising from about 35 mol % to about 45 mol % ofthe total lipid present in the particle; and (d) a PEG-lipid conjugatecomprising from about 5 mol % to about 10 mol % of the total lipidpresent in the particle, wherein at least about 95% of the particles inthe plurality of particles have a non-lamellar morphology. Thisembodiment of nucleic acid-lipid particle is generally referred toherein as the “7:54” formulation.

In still another specific embodiment, the composition of the presentinvention comprises: a plurality of nucleic acid-lipid particles,wherein each particle in the plurality of particles comprises: (a) anucleic acid (e.g., an interfering RNA); (b) a cationic lipid comprisingfrom about 55 mol % to about 65 mol % of the total lipid present in theparticle; (c) cholesterol or a derivative thereof comprising from about30 mol % to about 40 mol % of the total lipid present in the particle;and (d) a PEG-lipid conjugate comprising from about 5 mol % to about 10mol % of the total lipid present in the particle, wherein at least about95% of the particles in the plurality of particles have a non-lamellarmorphology. This embodiment of nucleic acid-lipid particle is generallyreferred to herein as the “7:58” formulation.

The present invention also provides a pharmaceutical compositioncomprising a composition of a plurality of nucleic acid lipid particles(e.g., SNALP), wherein at least about 95% of the particles in theplurality of particles have a non-lamellar morphology as describedherein and a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides a method forintroducing one or more active agents or therapeutic agents (e.g.,nucleic acid) into a cell, comprising contacting the cell with acomposition comprising a plurality of nucleic acid lipid particles(e.g., SNALP), wherein at least about 95% of the particles in theplurality of particles have a non-lamellar morphology as describedherein. In one embodiment, the cell is in a mammal and the mammal is ahuman. In another embodiment, the present invention provides a methodfor the in vivo delivery of one or more active agents or therapeuticagents (e.g., nucleic acid), comprising administering to a mammaliansubject a a composition comprising a plurality of nucleic acid lipidparticles (e.g., SNALP), wherein at least about 95% of the particles inthe plurality of particles have a non-lamellar morphology as describedherein. In a preferred embodiment, the mode of administration includes,but is not limited to, oral, intranasal, intravenous, intraperitoneal,intramuscular, intra-articular, intralesional, intratracheal,subcutaneous, and intradermal. Preferably, the mammalian subject is ahuman.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic of the Stepwise Dilution Method (SDM) or,alternatively, Lipomixer I method, used to make SNALP formulations. FIG.1B illustrates a schematic of the Direct Dilution Method (DDM) or,alternatively, the Lipomixer II method used to make SNALP formulations.

FIG. 2A-2C illustrates a schematic of an instrument used to carry outthe Cryo-Transmission Electron Microscopy analysis of various SNALPformulations and the cryo vetrification technique employed.

FIG. 3 shows Cryo-TEM data for the siApoB-8 10:15 SNALP formulationprepared by the Stepwise Dilution Method.

FIG. 4 shows Cryo-TEM data for the siApoB-8 2:30 SNALP formulationprepared by the Stepwise Dilution Method.

FIG. 5 shows Cryo-TEM data for the siApoB-8 1:57 SNALP formulationprepared by the Stepwise Dilution Method.

FIG. 6 shows Cryo-TEM data for the siApoB-8 1:62 SNALP formulationprepared by the Stepwise Dilution Method.

FIG. 7 shows the summary of particle morphology (non-lamellar particlesvs, lamellar particles) for the 10:15, the 2:30, the 1:57 and the 1:62formulations, all of which were prepared using the Stepwise DilutionMethod.

FIG. 8 shows Cryo-TEM data for the siApoB-8 2:30 SNALP formulationprepared by the Direct Dilution Method.

FIG. 9 shows Cryo-TEM data for the siApoB-8 2:40 SNALP formulationprepared by the Direct Dilution Method.

FIG. 10 shows Cryo-TEM data for the siApoB-8 1:57 SNALP formulationprepared by the Direct Dilution Method.

FIG. 11 shows Cryo-TEM data for the siApoB-8 1:62 SNALP formulationprepared by the Direct Dilution Method.

FIG. 12 shows the summary of particle morphology (non-lamellar particlesvs, lamellar particles) for the 10:15, the 2:30, the 1:57 and the 1:62formulations, all of which were prepared using the Direct DilutionMethod.

FIG. 13 shows Cryo-TEM data for the 7:54 PEG₇₅₀-C-DMA PLK-1 SNALPFormulation.

FIG. 14 shows Cryo-TEM data for the 7:54 PEG₇₅₀-C-DMA (−25% SNALP) PLK-1SNALP Formulation.

FIG. 15 shows Cryo-TEM data for the 7:54 PEG₇₅₀-C-DMA (+50% SNALP) PLK-1SNALP Formulation.

FIG. 16 illustrates data demonstrating that the 2:40 siApoB-8 SNALPformulation has enhanced ApoB silencing activity compared to the 2:30siApoB-8 SNALP formulation following intravenous administration in mice.

FIG. 17 illustrates data demonstrating the activity of 1:57 SNALPcontaining ApoB siRNA following intravenous administration in mice. Eachbar represents the group mean of five animals. Error bars indicate thestandard deviation.

FIG. 18 illustrates data demonstrating that the 1:57 siApoB-8 SNALPformulation has enhanced ApoB silencing activity compared to the 2:40siApoB-8 SNALP formulation following intravenous administration in mice.

FIG. 19 shows a comparison of the silencing activity of exemplary 1:57and 7:54 DLinDMA SNALP formulations in normal liver tissue and livertumors.

FIG. 20 shows a comparison of the silencing activity of exemplary 1:57and 7:54 DLinDMA SNALP formulations in subcutaneous tumors.

FIG. 21 illustrates an inverse Hexagonal (H₁₁) or Cubic Phase structureof the non-lamellar stable nucleic acid-lipid particles prepared by theDirect Dilution Method.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based, in part, upon the surprising discoverythat by controlling the lipid composition of a SNALP formulation as wellas the formation process used to prepare the SNALP formulation, a novelnon-lamellar lipid nanoparticle (i.e., SNALP) can be produced. Moreparticularly, it has surprisingly been found that lipid particles thatcomprise from about 50 mol % to about 85 mol % of a cationic lipid, fromabout 13 mol % to about 49.5 mol % of a non-cationic lipid, and fromabout 0.5 mol % to about 10 mol % of a lipid conjugate, and that aremade using the Direct Dilution Method as described herein have a novelnon-lamellar (i.e., non-bilayer) morphology and enhanced silencingability when used to deliver an interfering nucleic acid, such as ansiRNA molecule. As such, the present invention provides a compositioncomprising a plurality of nucleic acid-lipid particles, wherein eachparticle in the plurality of particles comprises: (a) a nucleic acid;(b) a cationic lipid comprising from about 50 mol % to about 85 mol % ofthe total lipid present in the particle; (c) a non-cationic lipidcomprising from about 13 mol % to about 49.5 mol % of the total lipidpresent in the particle; and (d) a conjugated lipid that inhibitsaggregation of particles comprising from about 0.5 mol % to about 10 mol% of the total lipid present in the particle, wherein at least about 95%of the particles in the plurality of particles have a non-lamellarmorphology. In preferred embodiments, greater than 95%, preferably,greater than 96%, preferably, greater than 97%, preferably, greater than98% and, preferably, greater than 99% of the particles have anon-lamellar morphology, i.e., a non-bilayer structure.

As illustrated in FIG. 21 and without intending to be bound by anytheory, it is believed that the cationic lipid and cholesterol combinetogether with the nucleic acid to form inverted micelle aggregates,wherein the nucleic acid is encapsulated by a membrane composed ofcationic lipid and cholesterol present in the formulation and,thereafter, spontaneous aggregation of the inverted micelles results incolloidal particles that are stabilized by the lipid surfactants (e.g.,the PEG-lipids and/or phospholipids present in the formulation). It isthought that the resulting non-lamellar (i.e., non-bilayer structure)particle has an an inverse Hexagonal (H₁₁) or Cubic phase structure. Inessence, it is thought that the resulting non-bilayer lipid packingprovides a 3-dimensional network of lipid cylinders with water andnucleic on the inside, i.e., essentially, a lipid dropletinterpenetrated with aqueous channels containg the nucleic acid.

The non-lamellar morphology (i.e., non-bilayer structure) of theresulting lipid particles can readily be determined using techniquesknown to and used by those of skill in the art. Such techniques include,but are not limited to, Cryo-Transmission Electron Microscopy(“Cryo-TEM”), Differential Scanning calorimetry (“DSC”), X-RayDiffraction, etc. As illustrated in FIGS. 3-6 and 8-11, the morphologyof the lipid particles (lamellar vs. non-lamellar) can readily beassessed and characterized using, e.g., Cryo-TEM analysis as describedherein.

It has been found that the SNALP of the present invention provideadvantages when used for the in vitro or in vivo delivery of an activeagent, such as a therapeutic nucleic acid (e.g., an interfering RNA). Inparticular, as illustrated by the Examples herein, the present inventionprovides stable nucleic acid-lipid particles (SNALP) that advantageouslyimpart increased activity of the encapsulated nucleic acid (e.g., aninterfering RNA such as siRNA) and improved tolerability of theformulations in vivo, resulting in a significant increase in thetherapeutic index as compared to nucleic acid-lipid particlecompositions previously described. Additionally, the SNALP of theinvention are stable in circulation, e.g., resistant to degradation bynucleases in serum, and are substantially non-toxic to mammals such ashumans. As a non-limiting example, FIG. 17 of Example 5 shows that oneSNALP embodiment of the invention (“1:57 SNALP”) was more efficacious ascompared to a nucleic acid-lipid particle previously described (“2:30SNALP”) in mediating target gene silencing at a 10-fold lower dose.Similarly, FIG. 18 of Example 6 shows that the “1:57 SNALP” formulationwas substantially more effective at silencing the expression of a targetgene as compared to nucleic acid-lipid particles previously described(“2:40 SNALP”). Moreover, FIG. 20 of Example 8 shows that the “7:54SNALP” PLK-1 displayed increased potency in SC tumors and that thisformulation can be used to preferentially target tumors outside of theliver.

In certain embodiments, the present invention provides improvedcompositions for the delivery of interfering RNA such as siRNAmolecules. In particular, the Examples herein illustrate that theimproved lipid particle formulations of the invention are highlyeffective in downregulating the mRNA and/or protein levels of targetgenes. Furthermore, the Examples herein illustrate that the presence ofcertain molar ratios of lipid component is results in improved orenhanced activity of these lipid particle formulations of the presentinvention. For instance, the “1:57 SNALP,” “1:62 SNALP,” “7:54 SNALP”and “7:58 SNALP” formulations described herein are exemplaryformulations of the present invention that are particularly advantageousbecause they provide improved efficacy and tolerability in vivo, areserum-stable, are are substantially non-toxic, are smaller in size, withsmaller polydispersities, are capable of accessing extravascular sites,and are capable of reaching target cell populations.

The lipid particles and compositions of the present invention may beused for a variety of purposes, including the delivery of associated orencapsulated therapeutic agents to cells, both in vitro and in vivo.Accordingly, the present invention provides methods for treatingdiseases or disorders in a subject in need thereof, by contacting thesubject with a lipid particle described herein comprising one or moresuitable therapeutic agents.

Various exemplary embodiments of the lipid particles of the invention,as well as compositions and formulations comprising the same, and theiruse to deliver therapeutic agents and modulate target gene and proteinexpression, are described in further detail below.

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The term “interfering RNA” or “RNAi” or “interfering RNA sequence” asused herein includes single-stranded RNA (e.g., mature miRNA, ssRNAioligonucleotides, ssDNAi oligonucleotides), double-stranded RNA (i.e.,duplex RNA such as siRNA, Dicer-substrate dsRNA, shRNA, aiRNA, orpre-miRNA), a DNA-RNA hybrid (see, e.g., PCT Publication No. WO2004/078941), or a DNA-DNA hybrid (see, e.g., PCT Publication No. WO2004/104199) that is capable of reducing or inhibiting the expression ofa target gene or sequence (e.g., by mediating the degradation orinhibiting the translation of mRNAs which are complementary to theinterfering RNA sequence) when the interfering RNA is in the same cellas the target gene or sequence. Interfering RNA thus refers to thesingle-stranded RNA that is complementary to a target mRNA sequence orto the double-stranded RNA formed by two complementary strands or by asingle, self-complementary strand. Interfering RNA may have substantialor complete identity to the target gene or sequence, or may comprise aregion of mismatch (i.e., a mismatch motif). The sequence of theinterfering RNA can correspond to the full-length target gene, or asubsequence thereof. Preferably, the interfering RNA molecules arechemically synthesized. The disclosures of each of the above patentdocuments are herein incorporated by reference in their entirety for allpurposes.

Interfering RNA includes “small-interfering RNA” or “siRNA,” e.g.,interfering RNA of about 15-60, 15-50, or 15-40 (duplex) nucleotides inlength, more typically about 15-30, 15-25, or 19-25 (duplex) nucleotidesin length, and is preferably about 20-24, 21-22, or 21-23 (duplex)nucleotides in length (e.g., each complementary sequence of thedouble-stranded siRNA is 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25nucleotides in length, preferably about 20-24, 21-22, or 21-23nucleotides in length, and the double-stranded siRNA is about 15-60,15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length, preferablyabout 18-22, 19-20, or 19-21 base pairs in length). siRNA duplexes maycomprise 3′ overhangs of about 1 to about 4 nucleotides or about 2 toabout 3 nucleotides and 5′ phosphate termini. Examples of siRNA include,without limitation, a double-stranded polynucleotide molecule assembledfrom two separate stranded molecules, wherein one strand is the sensestrand and the other is the complementary antisense strand; adouble-stranded polynucleotide molecule assembled from a single strandedmolecule, where the sense and antisense regions are linked by a nucleicacid-based or non-nucleic acid-based linker; a double-strandedpolynucleotide molecule with a hairpin secondary structure havingself-complementary sense and antisense regions; and a circularsingle-stranded polynucleotide molecule with two or more loop structuresand a stem having self-complementary sense and antisense regions, wherethe circular polynucleotide can be processed in vivo or in vitro togenerate an active double-stranded siRNA molecule. As used herein, theterm “siRNA” includes RNA-RNA duplexes as well as DNA-RNA hybrids (see,e.g., PCT Publication No. WO 2004/078941).

Preferably, siRNA are chemically synthesized. siRNA can also begenerated by cleavage of longer dsRNA (e.g., dsRNA greater than about 25nucleotides in length) with the E. coli RNase III or Dicer. Theseenzymes process the dsRNA into biologically active siRNA (see, e.g.,Yang et al., Proc. Natl. Acad. Sci. USA, 99:9942-9947 (2002); Calegariet al., Proc. Natl. Acad. Sci. USA, 99:14236 (2002); Byrom et al.,Ambion TechNotes, 10(1):4-6 (2003); Kawasaki et al., Nucleic Acids Res.,31:981-987 (2003); Knight et al., Science, 293:2269-2271 (2001); andRobertson et al., J. Biol. Chem., 243:82 (1968)). Preferably, dsRNA areat least 50 nucleotides to about 100, 200, 300, 400, or 500 nucleotidesin length. A dsRNA may be as long as 1000, 1500, 2000, 5000 nucleotidesin length, or longer. The dsRNA can encode for an entire gene transcriptor a partial gene transcript. In certain instances, siRNA may be encodedby a plasmid (e.g., transcribed as sequences that automatically foldinto duplexes with hairpin loops).

As used herein, the term “mismatch motif” or “mismatch region” refers toa portion of an interfering RNA (e.g., siRNA) sequence that does nothave 100% complementarity to its target sequence. An interfering RNA mayhave at least one, two, three, four, five, six, or more mismatchregions. The mismatch regions may be contiguous or may be separated by1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more nucleotides. The mismatchmotifs or regions may comprise a single nucleotide or may comprise two,three, four, five, or more nucleotides.

The phrase “inhibiting expression of a target gene” refers to theability of an interfering RNA (e.g., siRNA) to silence, reduce, orinhibit the expression of a target gene (e.g., PLK-1). To examine theextent of gene silencing, a test sample (e.g., a sample of cells inculture expressing the target gene) or a test mammal (e.g., a mammalsuch as a human or an animal model such as a rodent (e.g., mouse) or anon-human primate (e.g., monkey) model) is contacted with an interferingRNA (e.g., siRNA) that silences, reduces, or inhibits expression of thetarget gene. Expression of the target gene in the test sample or testanimal is compared to expression of the target gene in a control sample(e.g., a sample of cells in culture expressing the target gene) or acontrol mammal (e.g., a mammal such as a human or an animal model suchas a rodent (e.g., mouse) or non-human primate (e.g., monkey) model)that is not contacted with or administered the interfering RNA (e.g.,siRNA). The expression of the target gene in a control sample or acontrol mammal may be assigned a value of 100%. In particularembodiments, silencing, inhibition, or reduction of expression of atarget gene is achieved when the level of target gene expression in thetest sample or the test mammal relative to the level of target geneexpression in the control sample or the control mammal is about 95%,90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%,20%, 15%, 10%, 5%, or 0%. In other words, the interfering RNAs (e.g.,siRNAs) of the present invention are capable of silencing, reducing, orinhibiting the expression of a target gene (e.g., PLK-1) by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, or 100% in a test sample or a test mammalrelative to the level of target gene expression in a control sample or acontrol mammal not contacted with or administered the interfering RNA.Suitable assays for determining the level of target gene expressioninclude, without limitation, examination of protein or mRNA levels usingtechniques known to those of skill in the art, such as, e.g., dot blots,Northern blots, in situ hybridization, ELISA, immunoprecipitation,enzyme function, as well as phenotypic assays known to those of skill inthe art.

An “effective amount” or “therapeutically effective amount” of an activeagent or therapeutic agent such as an interfering RNA is an amountsufficient to produce the desired effect, e.g., an inhibition ofexpression of a target sequence in comparison to the normal expressionlevel detected in the absence of an interfering RNA. Inhibition ofexpression of a target gene or target sequence is achieved when thevalue obtained with an interfering RNA relative to the control is about95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%,25%, 20%, 15%, 10%, 5%, or 0%. Suitable assays for measuring expressionof a target gene or target sequence include, e.g., examination ofprotein or RNA levels using techniques known to those of skill in theart such as dot blots, northern blots, in situ hybridization, ELISA,immunoprecipitation, enzyme function, as well as phenotypic assays knownto those of skill in the art.

By “decrease,” “decreasing,” “reduce,” or “reducing” of an immuneresponse by an interfering RNA is intended to mean a detectable decreaseof an immune response to a given interfering RNA (e.g., a modifiedinterfering RNA). The amount of decrease of an immune response by amodified interfering RNA may be determined relative to the level of animmune response in the presence of an unmodified interfering RNA. Adetectable decrease can be about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or morelower than the immune response detected in the presence of theunmodified interfering RNA. A decrease in the immune response tointerfering RNA is typically measured by a decrease in cytokineproduction (e.g., IFNγ, IFNα, TNFα, IL-6, IL-8, or IL-12) by a respondercell in vitro or a decrease in cytokine production in the sera of amammalian subject after administration of the interfering RNA.

As used herein, the term “responder cell” refers to a cell, preferably amammalian cell, that produces a detectable immune response whencontacted with an immunostimulatory interfering RNA such as anunmodified siRNA. Exemplary responder cells include, e.g., dendriticcells, macrophages, peripheral blood mononuclear cells (PBMCs),splenocytes, and the like. Detectable immune responses include, e.g.,production of cytokines or growth factors such as TNF-α, IFN-α, IFN-γ,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, TGF, andcombinations thereof. Detectable immune responses also include, e.g.,induction of interferon-induced protein with tetratricopeptide repeats 1(IFIT 1) mRNA.

“Substantial identity” refers to a sequence that hybridizes to areference sequence under stringent conditions, or to a sequence that hasa specified percent identity over a specified region of a referencesequence.

The phrase “stringent hybridization conditions” refers to conditionsunder which a nucleic acid will hybridize to its target sequence,typically in a complex mixture of nucleic acids, but to no othersequences. Stringent conditions are sequence-dependent and will bedifferent in different circumstances. Longer sequences hybridizespecifically at higher temperatures. An extensive guide to thehybridization of nucleic acids is found in Tijssen, Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic Probes,“Overview of principles of hybridization and the strategy of nucleicacid assays” (1993). Generally, stringent conditions are selected to beabout 5-10° C. lower than the thermal melting point (T_(m)) for thespecific sequence at a defined ionic strength pH. The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. For selective orspecific hybridization, a positive signal is at least two timesbackground, preferably 10 times background hybridization.

Exemplary stringent hybridization conditions can be as follows: 50%formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS,incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. ForPCR, a temperature of about 36° C. is typical for low stringencyamplification, although annealing temperatures may vary between about32° C. and 48° C. depending on primer length. For high stringency PCRamplification, a temperature of about 62° C. is typical, although highstringency annealing temperatures can range from about 50° C. to about65° C., depending on the primer length and specificity. Typical cycleconditions for both high and low stringency amplifications include adenaturation phase of 90° C.-95° C. for 30 sec.-2 min., an annealingphase lasting 30 sec.-2 min., and an extension phase of about 72° C. for1-2 min. Protocols and guidelines for low and high stringencyamplification reactions are provided, e.g., in Innis et al., PCRProtocols, A Guide to Methods and Applications, Academic Press, Inc.N.Y. (1990).

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides whichthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. A positive hybridization is at least twicebackground. Those of ordinary skill will readily recognize thatalternative hybridization and wash conditions can be utilized to provideconditions of similar stringency. Additional guidelines for determininghybridization parameters are provided in numerous references, e.g.,Current Protocols in Molecular Biology, Ausubel et al., eds.

The terms “substantially identical” or “substantial identity,” in thecontext of two or more nucleic acids, refer to two or more sequences orsubsequences that are the same or have a specified percentage ofnucleotides that are the same (i.e., at least about 60%, preferably atleast about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over aspecified region), when compared and aligned for maximum correspondenceover a comparison window, or designated region as measured using one ofthe following sequence comparison algorithms or by manual alignment andvisual inspection. This definition, when the context indicates, alsorefers analogously to the complement of a sequence. Preferably, thesubstantial identity exists over a region that is at least about 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window,” as used herein, includes reference to a segmentof any one of a number of contiguous positions selected from the groupconsisting of from about 5 to about 60, usually about 10 to about 45,more usually about 15 to about 30, in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by thehomology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.,48:443 (1970), by the search for similarity method of Pearson andLipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see, e.g., Current Protocols in Molecular Biology, Ausubelet al., eds. (1995 supplement)).

Non-limiting examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity are the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nuc. AcidsRes., 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.,215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with theparameters described herein, to determine percent sequence identity forthe nucleic acids of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/). Anotherexample is a global alignment algorithm for determining percent sequenceidentiy such as the Needleman-Wunsch algorithm for aligning protein ornucleotide (e.g., RNA) sequences.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA, 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide sequences would occur by chance. For example, a nucleicacid is considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.2, more preferably less than about0.01, and most preferably less than about 0.001.

The term “nucleic acid” as used herein refers to a polymer containing atleast two deoxyribonucleotides or ribonucleotides in either single- ordouble-stranded form and includes DNA, RNA, and hybrids thereof. DNA maybe in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNAduplexes, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC,artificial chromosomes), expression cassettes, chimeric sequences,chromosomal DNA, or derivatives and combinations of these groups. RNAmay be in the form of small interfering RNA (siRNA), Dicer-substratedsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA),microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), andcombinations thereof. Nucleic acids include nucleic acids containingknown nucleotide analogs or modified backbone residues or linkages,which are synthetic, naturally occurring, and non-naturally occurring,and which have similar binding properties as the reference nucleic acid.Examples of such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2′-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unlessspecifically limited, the term encompasses nucleic acids containingknown analogues of natural nucleotides that have similar bindingproperties as the reference nucleic acid. Unless otherwise indicated, aparticular nucleic acid sequence also implicitly encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions), alleles, orthologs, SNPs, and complementary sequences aswell as the sequence explicitly indicated. Specifically, degeneratecodon substitutions may be achieved by generating sequences in which thethird position of one or more selected (or all) codons is substitutedwith mixed-base and/or deoxyinosine residues (Batzer et al., NucleicAcid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base,and a phosphate group. Nucleotides are linked together through thephosphate groups. “Bases” include purines and pyrimidines, which furtherinclude natural compounds adenine, thymine, guanine, cytosine, uracil,inosine, and natural analogs, and synthetic derivatives of purines andpyrimidines, which include, but are not limited to, modifications whichplace new reactive groups such as, but not limited to, amines, alcohols,thiols, carboxylates, and alkylhalides.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequencethat comprises partial length or entire length coding sequencesnecessary for the production of a polypeptide or precursor polypeptide(e.g., PLK-1).

“Gene product,” as used herein, refers to a product of a gene such as anRNA transcript or a polypeptide.

The term “lipid” refers to a group of organic compounds that include,but are not limited to, esters of fatty acids and are characterized bybeing insoluble in water, but soluble in many organic solvents. They areusually divided into at least three classes: (1) “simple lipids,” whichinclude fats and oils as well as waxes; (2) “compound lipids,” whichinclude phospholipids and glycolipids; and (3) “derived lipids” such assteroids.

The term “lipid particle” includes a lipid formulation that can be usedto deliver an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA), to a target site of interest (e.g., cell,tissue, organ, and the like). In preferred embodiments, the lipidparticle of the invention is a nucleic acid-lipid particle, which istypically formed from a cationic lipid, a non-cationic lipid, andoptionally a conjugated lipid that prevents aggregation of the particle.In other preferred embodiments, the active agent or therapeutic agent,such as a nucleic acid, may be encapsulated in the lipid portion of theparticle, thereby protecting it from enzymatic degradation.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a particle made from lipids (e.g., acationic lipid, a non-cationic lipid, and optionally a conjugated lipidthat prevents aggregation of the particle), wherein the nucleic acid(e.g., an interfering RNA) is fully encapsulated within the lipid. Incertain instances, SNALP are extremely useful for systemic applications,as they can exhibit extended circulation lifetimes following intravenous(i.v.) injection, they can accumulate at distal sites (e.g., sitesphysically separated from the administration site), and they can mediatesilencing of target gene expression at these distal sites. The nucleicacid may be complexed with a condensing agent and encapsulated within aSNALP as set forth in PCT Publication No. WO 00/03683, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes.

The lipid particles of the invention (e.g., SNALP) typically have a meandiameter of from about 30 nm to about 150 nm, from about 40 nm to about150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm,from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, fromabout 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm,and are substantially non-toxic. In addition, nucleic acids, whenpresent in the lipid particles of the present invention, are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Patent Publication Nos. 20040142025 and 20070042031, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

As used herein, “lipid encapsulated” can refer to a lipid particle thatprovides an active agent or therapeutic agent, such as a nucleic acid(e.g., an interfering RNA that targets PLK-1), with full encapsulation,partial encapsulation, or both. In a preferred embodiment, the nucleicacid is fully encapsulated in the lipid particle (e.g., to form a SNALPor other nucleic acid-lipid particle).

The term “lipid conjugate” refers to a conjugated lipid that inhibitsaggregation of lipid particles. Such lipid conjugates include, but arenot limited to, PEG-lipid conjugates such as, e.g., PEG coupled todialkyloxypropyls (e.g., PEG-DAA conjugates), PEG coupled todiacylglycerols (e.g., PEG-DAG conjugates), PEG coupled to cholesterol,PEG coupled to phosphatidylethanolamines, and PEG conjugated toceramides (see, e.g., U.S. Pat. No. 5,885,613), cationic PEG lipids,polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates; see,e.g., U.S. Provisional Application No. 61/294,828, filed Jan. 13, 2010,and U.S. Provisional Application No. 61/295,140, filed Jan. 14, 2010),polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof.Additional examples of POZ-lipid conjugates are described in PCTPublication No. WO 2010/006282. PEG or POZ can be conjugated directly tothe lipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG or the POZ to a lipid can be usedincluding, e.g., non-ester containing linker moieties andester-containing linker moieties. In certain preferred embodiments,non-ester containing linker moieties, such as amides or carbamates, areused. The disclosures of each of the above patent documents are hereinincorporated by reference in their entirety for all purposes.

The term “amphipathic lipid” refers, in part, to any suitable materialwherein the hydrophobic portion of the lipid material orients into ahydrophobic phase, while the hydrophilic portion orients toward theaqueous phase. Hydrophilic characteristics derive from the presence ofpolar or charged groups such as carbohydrates, phosphate, carboxylic,sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups.Hydrophobicity can be conferred by the inclusion of apolar groups thatinclude, but are not limited to, long-chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic, or heterocyclic group(s). Examples ofamphipathic compounds include, but are not limited to, phospholipids,aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limitedto, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine, anddilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus,such as sphingolipid, glycosphingolipid families, diacylglycerols, andβ-acyloxyacids, are also within the group designated as amphipathiclipids. Additionally, the amphipathic lipids described above can bemixed with other lipids including triglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid species thatexist either in an uncharged or neutral zwitterionic form at a selectedpH. At physiological pH, such lipids include, for example,diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well asany other neutral lipid or anionic lipid.

The term “anionic lipid” refers to any lipid that is negatively chargedat physiological pH. These lipids include, but are not limited to,phosphatidylglycerols, cardiolipins, diacylphosphatidylserines,diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines,N-succinyl phosphatidylethanolamines,N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifyinggroups joined to neutral lipids.

The term “hydrophobic lipid” refers to compounds having apolar groupsthat include, but are not limited to, long-chain saturated andunsaturated aliphatic hydrocarbon groups and such groups optionallysubstituted by one or more aromatic, cycloaliphatic, or heterocyclicgroup(s). Suitable examples include, but are not limited to,diacylglycerol, dialkylglycerol, N—N-dialkylamino,1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

The term “fusogenic” refers to the ability of a lipid particle, such asa SNALP, to fuse with the membranes of a cell. The membranes can beeither the plasma membrane or membranes surrounding organelles, e.g.,endosome, nucleus, etc.

The term “non-lamellar morphology” refer to a non-bilayer structure. Thenon-bilayer morphology can include, for example, three dimensionaltubes, rods, cubic symmetries, etc. The non-lamellar morphology (i.e.,non-bilayer structure) of the lipid particles disclosed herein can bedetermined using analytical techniques including Cryo-TransmissionElectron Microscopy (“Cryo-TEM”), Differential Scanning calorimetry(“DSC”), X-Ray Diffraction, etc.

The term “a plurality of nucleic acid-lipid particles” refers to atleast 2 particles, more preferably more than 10, 10², 10³, 10⁴, 10⁵, 10⁶or more particles (or any fraction thereof or range therein). In certainembodiments, the plurality of nucleic acid-lipid particles includes50-100, 50-200, 50-300, 50-400, 50-500, 50-600, 50-700, 50-800, 50-900,50-1000, 50-1100, 50-1200, 50-1300, 50-1400, 50-1500, 50-1600, 50-1700,50-1800, 50-1900, 50-2000, 50-2500, 50-3000, 50-3500, 50-4000, 50-4500,50-5000, 50-5500, 50-6000, 50-6500, 50-7000, 50-7500, 50-8000, 50-8500,50-9000, 50-9500, 50-10,000 or more particles. It will be apparent tothose of skill in the art that the plurality of nucleic acid-lipidparticles can include any fraction of the foregoing ranges or any rangetherein. In certain other embodiments, the plurality of nucleicacid-lipid particles is the number of particles (or a representativesubset of particles) observed in a Cryo-TEM image similar to thoseillustrated in FIGS. 3-6 and 8-11, wherein the Cryo-TEM analysis iscarried out using a method similar to that set forth in Example 2.

As used herein, the term “aqueous solution” refers to a compositioncomprising in whole, or in part, water.

As used herein, the term “organic lipid solution” refers to acomposition comprising in whole, or in part, an organic solvent having alipid.

“Distal site,” as used herein, refers to a physically separated site,which is not limited to an adjacent capillary bed, but includes sitesbroadly distributed throughout an organism.

“Serum-stable” in relation to nucleic acid-lipid particles such as SNALPmeans that the particle is not significantly degraded after exposure toa serum or nuclease assay that would significantly degrade free DNA orRNA. Suitable assays include, for example, a standard serum assay, aDNAse assay, or an RNAse assay.

“Systemic delivery,” as used herein, refers to delivery of lipidparticles that leads to a broad biodistribution of an active agent suchas an interfering RNA (e.g., siRNA) within an organism. Some techniquesof administration can lead to the systemic delivery of certain agents,but not others. Systemic delivery means that a useful, preferablytherapeutic, amount of an agent is exposed to most parts of the body. Toobtain broad biodistribution generally requires a blood lifetime suchthat the agent is not rapidly degraded or cleared (such as by first passorgans (liver, lung, etc.) or by rapid, nonspecific cell binding) beforereaching a disease site distal to the site of administration. Systemicdelivery of lipid particles can be by any means known in the artincluding, for example, intravenous, subcutaneous, and intraperitoneal.In a preferred embodiment, systemic delivery of lipid particles is byintravenous delivery.

“Local delivery,” as used herein, refers to delivery of an active agentsuch as an interfering RNA (e.g., siRNA) directly to a target sitewithin an organism. For example, an agent can be locally delivered bydirect injection into a disease site such as a tumor or other targetsite such as a site of inflammation or a target organ such as the liver,heart, pancreas, kidney, and the like.

The term “mammal” refers to any mammalian species such as a human,mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and thelike.

The term “cancer” refers to any member of a class of diseasescharacterized by the uncontrolled growth of aberrant cells. The termincludes all known cancers and neoplastic conditions, whethercharacterized as malignant, benign, soft tissue, or solid, and cancersof all stages and grades including pre- and post-metastatic cancers.Examples of different types of cancer include, but are not limited to,liver cancer, lung cancer, colon cancer, rectal cancer, anal cancer,bile duct cancer, small intestine cancer, stomach (gastric) cancer,esophageal cancer; gallbladder cancer, pancreatic cancer, appendixcancer, breast cancer, ovarian cancer; cervical cancer, prostate cancer,renal cancer (e.g., renal cell carcinoma), cancer of the central nervoussystem, glioblastoma, skin cancer, lymphomas, choriocarcinomas, head andneck cancers, osteogenic sarcomas, and blood cancers. Non-limitingexamples of specific types of liver cancer include hepatocellularcarcinoma (HCC), secondary liver cancer (e.g., caused by metastasis ofsome other non-liver cancer cell type), and hepatoblastoma. As usedherein, a “tumor” comprises one or more cancerous cells.

The term “polo-like kinase 1,” “PLK-1,” “polo-like kinase,” or “PLK”refers to a serine/threonine kinase containing two functional domains:(1) a kinase domain; and (2) a polo-box domain (see, e.g., Barr et al.,Nat. Rev. Mol. Cell Biol., 5:429-440 (2004)). The activity and cellularconcentration of PLK-1 are crucial for the precise regulation of celldivision. PLK-1 expression and activity are low throughout the G0, G1,and S phases of the cell cycle, but begin to rise in G2 and peak duringM phase. PLK-1 is essential for mitosis and cell division andcontributes to the following processes: centrosome maturation and theactivation of maturation-promoting factors by Cdc25C and cyclinB1phosphorylation; bipolar spindle formation; and DNA damage checkpointadaptation (DNA damage inhibits PLK-1 in G2 and mitosis). PLK-1 is alsoinvolved in the activation of components of the anaphase promotingcomplex for mitotic exit and cytokinesis. PLK-1 is overexpressed in manycancer types including hepatoma and colon cancer, and PLK-1 expressionoften correlates with poor patient prognosis. Overexpression of PLK-1(wild-type or kinase inactive) results in multinucleation (geneticinstability). Hyperactive PLK-1 overrides the DNA damage checkpoint.Constitutive PLK-1 expression causes transformation of NIH 3T3 cells.PLK-1 phosphorylates the p53 tumor suppressor, thereby inhibiting thepro-apoptotic effects of p53. Human PLK-1 mRNA sequences are set forthin Genbank Accession Nos. NM_005030, X73458, BC014846, BC003002,HSU01038, and L19559. A mouse PLK-1 mRNA sequence is set forth inGenbank Accession No. NM_011121. PLK-1 is also known as serine/threonineprotein kinase 13 (STPK13).

III. Description of the Embodiments

The present invention provides novel, serum-stable lipid particlescomprising one or more active agents or therapeutic agents, methods ofmaking the lipid particles, and methods of delivering and/oradministering the lipid particles (e.g., for the treatment of a diseaseor disorder).

In one aspect, the present invention provides a composition comprising aplurality of nucleic acid-lipid particles, wherein each particle in theplurality of particles comprises: (a) a nucleic acid; (b) a cationiclipid comprising from about 50 mol % to about 85 mol % of the totallipid present in the particle; (c) a non-cationic lipid comprising fromabout 13 mol % to about 49.5 mol % of the total lipid present in theparticle; and (d) a conjugated lipid that inhibits aggregation ofparticles comprising from about 0.5 mol % to about 10 mol % of the totallipid present in the particle, wherein at least about 95% of theparticles in the plurality of particles have a non-lamellar morphology.In preferred embodiments, greater than 95%, preferably, greater than96%, preferably, greater than 97%, preferably, greater than 98% and,preferably, greater than 99% of the particles have a non-lamellarmorphology, i.e., a non-bilayer structure.

In certain embodiments, the active agent or therapeutic agent is fullyencapsulated within the lipid portion of the lipid particles such thatthe active agent or therapeutic agent in the lipid particle is resistantin aqueous solution to enzymatic degradation, e.g., by a nuclease orprotease. In certain other embodiments, the lipid particles aresubstantially non-toxic to mammals such as humans.

In some embodiments, the active agent or therapeutic agent comprises anucleic acid. In certain instances, the nucleic acid comprises aninterfering RNA molecule such as, e.g., an siRNA, aiRNA, miRNA, ormixtures thereof. In certain other instances, the nucleic acid comprisessingle-stranded or double-stranded DNA, RNA, or a DNA/RNA hybrid suchas, e.g., an antisense oligonucleotide, a ribozyme, a plasmid, animmunostimulatory oligonucleotide, or mixtures thereof.

In other embodiments, the active agent or therapeutic agent comprises apeptide or polypeptide. In certain instances, the peptide or polypeptidecomprises an antibody such as, e.g., a polyclonal antibody, a monoclonalantibody, an antibody fragment; a humanized antibody, a recombinantantibody, a recombinant human antibody, a Primatized™ antibody, ormixtures thereof. In certain other instances, the peptide or polypeptidecomprises a cytokine, a growth factor, an apoptotic factor, adifferentiation-inducing factor, a cell-surface receptor, a ligand, ahormone, a small molecule (e.g., small organic molecule or compound), ormixtures thereof.

In preferred embodiments, the active agent or therapeutic agentcomprises an siRNA. In one embodiment, the siRNA molecule comprises adouble-stranded region of about 15 to about 60 nucleotides in length(e.g., about 15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 nucleotides inlength, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides inlength). The siRNA molecules of the invention are capable of silencingthe expression of a target sequence in vitro and/or in vivo.

In some embodiments, the siRNA molecule comprises at least one modifiednucleotide. In certain preferred embodiments, the siRNA moleculecomprises one, two, three, four, five, six, seven, eight, nine, ten, ormore modified nucleotides in the double-stranded region. In certaininstances, the siRNA comprises from about 1% to about 100% (e.g., about1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100%) modified nucleotides in thedouble-stranded region. In preferred embodiments, less than about 25%(e.g., less than about 25%, 20%, 15%, 10%, or 5%) or from about 1% toabout 25% (e.g., from about 1%-25%, 5%-25%, 10%-25%, 15%-25%, 20%-25%,or 10%-20%) of the nucleotides in the double-stranded region comprisemodified nucleotides.

In other embodiments, the siRNA molecule comprises modified nucleotidesincluding, but not limited to, 2′-O-methyl (2′OMe) nucleotides,2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, and mixtures thereof. In preferred embodiments, the siRNAcomprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosinenucleotides, and mixtures thereof. In certain instances, the siRNA doesnot comprise 2′OMe-cytosine nucleotides. In other embodiments, the siRNAcomprises a hairpin loop structure.

The siRNA may comprise modified nucleotides in one strand (i.e., senseor antisense) or both strands of the double-stranded region of the siRNAmolecule. Preferably, uridine and/or guanosine nucleotides are modifiedat selective positions in the double-stranded region of the siRNAduplex. With regard to uridine nucleotide modifications, at least one,two, three, four, five, six, or more of the uridine nucleotides in thesense and/or antisense strand can be a modified uridine nucleotide suchas a 2′OMe-uridine nucleotide. In some embodiments, every uridinenucleotide in the sense and/or antisense strand is a 2′OMe-uridinenucleotide. With regard to guanosine nucleotide modifications, at leastone, two, three, four, five, six, or more of the guanosine nucleotidesin the sense and/or antisense strand can be a modified guanosinenucleotide such as a 2′OMe-guanosine nucleotide. In some embodiments,every guanosine nucleotide in the sense and/or antisense strand is a2′OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six,seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified,e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/orby introducing modified nucleotides such as 2′OMe nucleotides. The5′-GU-3′ motif can be in the sense strand, the antisense strand, or bothstrands of the siRNA sequence. The 5′-GU-3′ motifs may be adjacent toeach other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more nucleotides.

In some preferred embodiments, a modified siRNA molecule is lessimmunostimulatory than a corresponding unmodified siRNA sequence. Insuch embodiments, the modified siRNA molecule with reducedimmunostimulatory properties advantageously retains RNAi activityagainst the target sequence. In another embodiment, theimmunostimulatory properties of the modified siRNA molecule and itsability to silence target gene expression can be balanced or optimizedby the introduction of minimal and selective 2′OMe modifications withinthe siRNA sequence such as, e.g., within the double-stranded region ofthe siRNA duplex. In certain instances, the modified siRNA is at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% less immunostimulatory than the corresponding unmodified siRNA. Itwill be readily apparent to those of skill in the art that theimmunostimulatory properties of the modified siRNA molecule and thecorresponding unmodified siRNA molecule can be determined by, forexample, measuring INF-α and/or IL-6 levels from about two to abouttwelve hours after systemic administration in a mammal or transfectionof a mammalian responder cell using an appropriate lipid-based deliverysystem (such as the SNALP delivery system disclosed herein).

In certain embodiments, a modified siRNA molecule has an IC50 (i.e.,half-maximal inhibitory concentration) less than or equal to ten-foldthat of the corresponding unmodified siRNA (i.e., the modified siRNA hasan IC50 that is less than or equal to ten-times the IC50 of thecorresponding unmodified siRNA). In other embodiments, the modifiedsiRNA has an IC50 less than or equal to three-fold that of thecorresponding unmodified siRNA sequence. In yet other embodiments, themodified siRNA has an IC50 less than or equal to two-fold that of thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that a dose-response curve can be generated and theIC50 values for the modified siRNA and the corresponding unmodifiedsiRNA can be readily determined using methods known to those of skill inthe art.

In yet another embodiment, a modified siRNA molecule is capable ofsilencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of theexpression of the target sequence relative to the correspondingunmodified siRNA sequence.

In some embodiments, the siRNA molecule does not comprise phosphatebackbone modifications, e.g., in the sense and/or antisense strand ofthe double-stranded region. In other embodiments, the siRNA comprisesone, two, three, four, or more phosphate backbone modifications, e.g.,in the sense and/or antisense strand of the double-stranded region. Inpreferred embodiments, the siRNA does not comprise phosphate backbonemodifications.

In further embodiments, the siRNA does not comprise 2′-deoxynucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region. In yet further embodiments, the siRNA comprisesone, two, three, four, or more 2′-deoxy nucleotides, e.g., in the senseand/or antisense strand of the double-stranded region. In preferredembodiments, the siRNA does not comprise 2′-deoxy nucleotides.

In certain instances, the nucleotide at the 3′-end of thedouble-stranded region in the sense and/or antisense strand is not amodified nucleotide. In certain other instances, the nucleotides nearthe 3′-end (e.g., within one, two, three, or four nucleotides of the3′-end) of the double-stranded region in the sense and/or antisensestrand are not modified nucleotides.

The siRNA molecules described herein may have 3′ overhangs of one, two,three, four, or more nucleotides on one or both sides of thedouble-stranded region, or may lack overhangs (i.e., have blunt ends) onone or both sides of the double-stranded region. Preferably, the siRNAhas 3′ overhangs of two nucleotides on each side of the double-strandedregion. In certain instances, the 3′ overhang on the antisense strandhas complementarity to the target sequence and the 3′ overhang on thesense strand has complementarity to a complementary strand of the targetsequence. Alternatively, the 3′ overhangs do not have complementarity tothe target sequence or the complementary strand thereof. In someembodiments, the 3′ overhangs comprise one, two, three, four, or morenucleotides such as 2′-deoxy (2′H) nucleotides. In certain preferredembodiments, the 3′ overhangs comprise deoxythymidine (dT) and/oruridine nucleotides. In other embodiments, one or more of thenucleotides in the 3′ overhangs on one or both sides of thedouble-stranded region comprise modified nucleotides. Non-limitingexamples of modified nucleotides are described above and include 2′OMenucleotides, 2′-deoxy-2′F nucleotides, 2′-deoxy nucleotides, 2′-O-2-MOEnucleotides, LNA nucleotides, and mixtures thereof. In preferredembodiments, one, two, three, four, or more nucleotides in the 3′overhangs present on the sense and/or antisense strand of the siRNAcomprise 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosinenucleotides, and mixtures thereof.

The siRNA may comprise at least one or a cocktail (e.g., at least two,three, four, five, six, seven, eight, nine, ten, or more) of unmodifiedand/or modified siRNA sequences that silence target gene expression. Thecocktail of siRNA may comprise sequences which are directed to the sameregion or domain (e.g., a “hot spot”) and/or to different regions ordomains of one or more target genes. In certain instances, one or more(e.g., at least two, three, four, five, six, seven, eight, nine, ten, ormore) modified siRNA that silence target gene expression are present ina cocktail. In certain other instances, one or more (e.g., at least two,three, four, five, six, seven, eight, nine, ten, or more) unmodifiedsiRNA sequences that silence target gene expression are present in acocktail.

In some embodiments, the antisense strand of the siRNA moleculecomprises or consists of a sequence that is at least about 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% complementary to the target sequence ora portion thereof. In other embodiments, the antisense strand of thesiRNA molecule comprises or consists of a sequence that is 100%complementary to the target sequence or a portion thereof. In furtherembodiments, the antisense strand of the siRNA molecule comprises orconsists of a sequence that specifically hybridizes to the targetsequence or a portion thereof.

In further embodiments, the sense strand of the siRNA molecule comprisesor consists of a sequence that is at least about 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the target sequence or a portionthereof. In additional embodiments, the sense strand of the siRNAmolecule comprises or consists of a sequence that is 100% identical tothe target sequence or a portion thereof.

In the lipid particles of the invention (e.g., SNALP comprising aninterfering RNA such as siRNA), the cationic lipid may comprise, e.g.,one or more of the following: the cationic lipids of Formula I asdisclosed herein, including, for example, MC3, LenMC3, CP-LenMC3,γ-LenMC3, CP-γ-LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide,Pan-MC3, Pan-MC4 and Pan MC5, 1,2-dilinoleyloxy-N,N-dimethylaminopropane(DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;“XTC2”), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)[1,3]-dioxolane(DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)[1,3]-dioxolane(DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane(DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane(DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane(DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane(DLin-C-DAP), 1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane(DLin-DAC), 1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), ormixtures thereof. In certain preferred embodiments, the cationic lipidis DLinDMA, DLin-K-C2-DMA (“XTC2”), MC3, LenMC3, CP-LenMC3, γ-LenMC3,CP-γ-LenMC3, MC3MC, MC2MC, MC3 Ether, MC4 Ether, MC3 Amide, Pan-MC3,Pan-MC4, Pan MC5, or mixtures thereof.

The synthesis of cationic lipids such as DLin-K-C2-DMA (“XTC2”),DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well asadditional cationic lipids, is described in U.S. Provisional ApplicationNo. 61/104,212, filed Oct. 9, 2008, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Thesynthesis of cationic lipids such as DLin-K-DMA, DLin-C-DAP, DLin-DAC,DLin-MA, DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl,DLin-MPZ, DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationiclipids, is described in PCT Application No. PCT/US08/88676, filed Dec.31, 2008, the disclosure of which is herein incorporated by reference inits entirety for all purposes. The synthesis of cationic lipids such asCLinDMA, as well as additional cationic lipids, is described in U.S.Patent Publication No. 20060240554, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

In some embodiments, the cationic lipid may comprise from about 50 mol %to about 90 mol %, from about 50 mol % to about 85 mol %, from about 50mol % to about 80 mol %, from about 50 mol % to about 75 mol %, fromabout 50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %,or from about 50 mol % to about 60 mol % of the total lipid present inthe particle.

In other embodiments, the cationic lipid may comprise from about 55 mol% to about 90 mol %, from about 55 mol % to about 85 mol %, from about55 mol % to about 80 mol %, from about 55 mol % to about 75 mol %, fromabout 55 mol % to about 70 mol %, or from about 55 mol % to about 65 mol% of the total lipid present in the particle.

In yet other embodiments, the cationic lipid may comprise from about 60mol % to about 90 mol %, from about 60 mol % to about 85 mol %, fromabout 60 mol % to about 80 mol %, from about 60 mol % to about 75 mol %,or from about 60 mol % to about 70 mol % of the total lipid present inthe particle.

In still yet other embodiments, the cationic lipid may comprise fromabout 65 mol % to about 90 mol %, from about 65 mol % to about 85 mol %,from about 65 mol % to about 80 mol %, or from about 65 mol % to about75 mol % of the total lipid present in the particle.

In further embodiments, the cationic lipid may comprise from about 70mol % to about 90 mol %, from about 70 mol % to about 85 mol %, fromabout 70 mol % to about 80 mol %, from about 75 mol % to about 90 mol %,from about 75 mol % to about 85 mol %, or from about 80 mol % to about90 mol % of the total lipid present in the particle.

In additional embodiments, the cationic lipid may comprise (at least)about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,84, 85, 86, 87, 88, 89, or 90 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In the lipid particles of the invention (e.g., SNALP comprising aninterfering RNA such as siRNA), the non-cationic lipid may comprise,e.g., one or more anionic lipids and/or neutral lipids. In preferredembodiments, the non-cationic lipid comprises one of the followingneutral lipid components: (1) cholesterol or a derivative thereof; (2) aphospholipid; or (3) a mixture of a phospholipid and cholesterol or aderivative thereof.

Examples of cholesterol derivatives include, but are not limited to,cholestanol, cholestanone, cholestenone, coprostanol,cholesteryl-2′-hydroxyethyl ether, cholesteryl-4′-hydroxybutyl ether,and mixtures thereof. The synthesis of cholesteryl-2′-hydroxyethyl etheris described herein.

The phospholipid may be a neutral lipid including, but not limited to,dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), egg phosphatidylcholine(EPC), and mixtures thereof. In certain preferred embodiments, thephospholipid is DPPC, DSPC, or mixtures thereof.

In some embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 60 mol %, from about 15 mol % to about 60 mol %, from about 20 mol% to about 60 mol %, from about 25 mol % to about 60 mol %, from about30 mol % to about 60 mol %, from about 10 mol % to about 55 mol %, fromabout 15 mol % to about 55 mol %, from about 20 mol % to about 55 mol %,from about 25 mol % to about 55 mol %, from about 30 mol % to about 55mol %, from about 13 mol % to about 50 mol %, from about 15 mol % toabout 50 mol % or from about 20 mol % to about 50 mol % of the totallipid present in the particle. When the non-cationic lipid is a mixtureof a phospholipid and cholesterol or a cholesterol derivative, themixture may comprise up to about 40, 50, or 60 mol % of the total lipidpresent in the particle.

In other embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 49.5 mol %, from about 13 mol % to about 49.5 mol %, from about 15mol % to about 49.5 mol %, from about 20 mol % to about 49.5 mol %, fromabout 25 mol % to about 49.5 mol %, from about 30 mol % to about 49.5mol %, from about 35 mol % to about 49.5 mol %, or from about 40 mol %to about 49.5 mol % of the total lipid present in the particle.

In yet other embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 45 mol %, from about 13 mol % to about 45 mol %, from about 15 mol% to about 45 mol %, from about 20 mol % to about 45 mol %, from about25 mol % to about 45 mol %, from about 30 mol % to about 45 mol %, orfrom about 35 mol % to about 45 mol % of the total lipid present in theparticle.

In still yet other embodiments, the non-cationic lipid (e.g., one ormore phospholipids and/or cholesterol) may comprise from about 10 mol %to about 40 mol %, from about 13 mol % to about 40 mol %, from about 15mol % to about 40 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 40 mol %, or from about 30 mol % to about 40 mol% of the total lipid present in the particle.

In further embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 35 mol %, from about 13 mol % to about 35 mol %, from about 15 mol% to about 35 mol %, from about 20 mol % to about 35 mol %, or fromabout 25 mol % to about 35 mol % of the total lipid present in theparticle.

In yet further embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise from about 10 mol % toabout 30 mol %, from about 13 mol % to about 30 mol %, from about 15 mol% to about 30 mol %, from about 20 mol % to about 30 mol %, from about10 mol % to about 25 mol %, from about 13 mol % to about 25 mol %, orfrom about 15 mol % to about 25 mol % of the total lipid present in theparticle.

In additional embodiments, the non-cationic lipid (e.g., one or morephospholipids and/or cholesterol) may comprise (at least) about 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle.

In certain preferred embodiments, the non-cationic lipid comprisescholesterol or a derivative thereof of from about 31.5 mol % to about42.5 mol % of the total lipid present in the particle. As a non-limitingexample, a phospholipid-free lipid particle of the invention maycomprise cholesterol or a derivative thereof at about 37 mol % of thetotal lipid present in the particle. In other preferred embodiments, aphospholipid-free lipid particle of the invention may comprisecholesterol or a derivative thereof of from about 30 mol % to about 45mol %, from about 30 mol % to about 40 mol %, from about 30 mol % toabout 35 mol %, from about 35 mol % to about 45 mol %, from about 40 mol% to about 45 mol %, from about 32 mol % to about 45 mol %, from about32 mol % to about 42 mol %, from about 32 mol % to about 40 mol %, fromabout 34 mol % to about 45 mol %, from about 34 mol % to about 42 mol %,from about 34 mol % to about 40 mol %, or about 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 mol % (or any fraction thereofor range therein) of the total lipid present in the particle.

In certain other preferred embodiments, the non-cationic lipid comprisesa mixture of: (i) a phospholipid of from about 4 mol % to about 10 mol %of the total lipid present in the particle; and (ii) cholesterol or aderivative thereof of from about 30 mol % to about 40 mol % of the totallipid present in the particle. As a non-limiting example, a lipidparticle comprising a mixture of a phospholipid and cholesterol maycomprise DPPC at about 7 mol % and cholesterol at about 34 mol % of thetotal lipid present in the particle. In other embodiments, thenon-cationic lipid comprises a mixture of: (i) a phospholipid of fromabout 3 mol % to about 15 mol %, from about 4 mol % to about 15 mol %,from about 4 mol % to about 12 mol %, from about 4 mol % to about 10 mol%, from about 4 mol % to about 8 mol %, from about 5 mol % to about 12mol %, from about 5 mol % to about 9 mol %, from about 6 mol % to about12 mol %, from about 6 mol % to about 10 mol %, or about 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, or 15 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle; and (ii)cholesterol or a derivative thereof of from about 25 mol % to about 45mol %, from about 30 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 40 mol %, from about 25 mol% to about 35 mol %, from about 30 mol % to about 35 mol %, from about35 mol % to about 45 mol %, from about 40 mol % to about 45 mol %, fromabout 28 mol % to about 40 mol %, from about 28 mol % to about 38 mol %,from about 30 mol % to about 38 mol %, from about 32 mol % to about 36mol %, or about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In further preferred embodiments, the non-cationic lipid comprises amixture of: (i) a phospholipid of from about 10 mol % to about 30 mol %of the total lipid present in the particle; and (ii) cholesterol or aderivative thereof of from about 10 mol % to about 30 mol % of the totallipid present in the particle. As a non-limiting example, a lipidparticle comprising a mixture of a phospholipid and cholesterol maycomprise DPPC at about 20 mol % and cholesterol at about 20 mol % of thetotal lipid present in the particle. In other embodiments, thenon-cationic lipid comprises a mixture of: (i) a phospholipid of fromabout 10 mol % to about 30 mol %, from about 10 mol % to about 25 mol %,from about 10 mol % to about 20 mol %, from about 15 mol % to about 30mol %, from about 20 mol % to about 30 mol %, from about 15 mol % toabout 25 mol %, from about 12 mol % to about 28 mol %, from about 14 mol% to about 26 mol %, or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mol % (or any fractionthereof or range therein) of the total lipid present in the particle;and (ii) cholesterol or a derivative thereof of from about 10 mol % toabout 30 mol %, from about 10 mol % to about 25 mol %, from about 10 mol% to about 20 mol %, from about 15 mol % to about 30 mol %, from about20 mol % to about 30 mol %, from about 15 mol % to about 25 mol %, fromabout 12 mol % to about 28 mol %, from about 14 mol % to about 26 mol %,or about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30 mol % (or any fraction thereof or range therein)of the total lipid present in the particle.

In the lipid particles of the invention (e.g., SNALP comprising aninterfering RNA such as siRNA), the conjugated lipid that inhibitsaggregation of particles may comprise, e.g., one or more of thefollowing: a polyethyleneglycol (PEG)-lipid conjugate, a polyamide(ATTA)-lipid conjugate, a cationic-polymer-lipid conjugates (CPLs), ormixtures thereof. In one preferred embodiment, the nucleic acid-lipidparticles comprise either a PEG-lipid conjugate or an ATTA-lipidconjugate. In certain embodiments, the PEG-lipid conjugate or ATTA-lipidconjugate is used together with a CPL. The conjugated lipid thatinhibits aggregation of particles may comprise a PEG-lipid including,e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEG-DAAconjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl(C14), a PEG-dipalmityloxypropyl (C16), a PEG-distearyloxypropyl (C18),or mixtures thereof.

Additional PEG-lipid conjugates suitable for use in the inventioninclude, but are not limited to,mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). Thesynthesis of PEG-C-DOMG is described in PCT Application No.PCT/US08/88676, filed Dec. 31, 2008, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Yetadditional PEG-lipid conjugates suitable for use in the inventioninclude, without limitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-w-methyl-poly(ethyleneglycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S.Pat. No. 7,404,969, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In some embodiments, the conjugated lipid that inhibits aggregation ofparticles is a CPL that has the formula: A-W—Y, wherein A is a lipidmoiety, W is a hydrophilic polymer, and Y is a polycationic moiety. Wmay be a polymer selected from the group consisting ofpolyethyleneglycol (PEG), polyamide, polylactic acid, polyglycolic acid,polylactic acid/polyglycolic acid copolymers, or combinations thereof,the polymer having a molecular weight of from about 250 to about 7000daltons. In some embodiments, Y has at least 4 positive charges at aselected pH. In some embodiments, Y may be lysine, arginine, asparagine,glutamine, derivatives thereof, or combinations thereof.

In certain instances, such as with the “1:57 SNALP” and “1:62 SNALP”formulations, the conjugated lipid that inhibits aggregation ofparticles (e.g., PEG-lipid conjugate) may comprise from about 0.1 mol %to about 2 mol %, from about 0.5 mol % to about 2 mol %, from about 1mol % to about 2 mol %, from about 0.6 mol % to about 1.9 mol %, fromabout 0.7 mol % to about 1.8 mol %, from about 0.8 mol % to about 1.7mol %, from about 1 mol % to about 1.8 mol %, from about 1.2 mol % toabout 1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3mol % to about 1.6 mol %, from about 1.4 mol % to about 1.5 mol %, orabout 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. Typically, in such instances, the PEG moiety has an averagemolecular weight of about 2,000 daltons.

In certain other instances, such as with the “7:54 SNALP” and “7:58SNALP” formulations, the conjugated lipid that inhibits aggregation ofparticles (e.g., PEG-lipid conjugate) may comprise from about 5.0 mol %to about 10 mol %, from about 5 mol % to about 9 mol %, from about 5 mol% to about 8 mol %, from about 6 mol % to about 9 mol %, from about 6mol % to about 8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9mol %, or 10 mol % (or any fraction thereof or range therein) of thetotal lipid present in the particle. Typically, in such instances, thePEG moiety has an average molecular weight of about 750 daltons.

In the lipid particles of the invention, the active agent or therapeuticagent may be fully encapsulated within the lipid portion of theparticle, thereby protecting the active agent or therapeutic agent fromenzymatic degradation. In preferred embodiments, a SNALP comprising anucleic acid such as an interfering RNA (e.g., siRNA) is fullyencapsulated within the lipid portion of the particle, therebyprotecting the nucleic acid from nuclease degradation. In certaininstances, the nucleic acid in the SNALP is not substantially degradedafter exposure of the particle to a nuclease at 37° C. for at leastabout 20, 30, 45, or 60 minutes. In certain other instances, the nucleicacid in the SNALP is not substantially degraded after incubation of theparticle in serum at 37° C. for at least about 30, 45, or 60 minutes orat least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, or 36 hours. In other embodiments, the active agentor therapeutic agent (e.g., nucleic acid such as siRNA) is complexedwith the lipid portion of the particle. One of the benefits of theformulations of the present invention is that the lipid particlecompositions are substantially non-toxic to mammals such as humans.

The term “fully encapsulated” indicates that the active agent ortherapeutic agent in the lipid particle is not significantly degradedafter exposure to serum or a nuclease or protease assay that wouldsignificantly degrade free DNA, RNA, or protein. In a fully encapsulatedsystem, preferably less than about 25% of the active agent ortherapeutic agent in the particle is degraded in a treatment that wouldnormally degrade 100% of free active agent or therapeutic agent, morepreferably less than about 10%, and most preferably less than about 5%of the active agent or therapeutic agent in the particle is degraded. Inthe context of nucleic acid therapeutic agents, full encapsulation maybe determined by an Oligreen® assay. Oligreen® is an ultra-sensitivefluorescent nucleic acid stain for quantitating oligonucleotides andsingle-stranded DNA or RNA in solution (available from InvitrogenCorporation; Carlsbad, Calif.). “Fully encapsulated” also indicates thatthe lipid particles are serum-stable, that is, that they do not rapidlydecompose into their component parts upon in vivo administration.

In another aspect, the present invention provides a lipid particle(e.g., SNALP) composition comprising a plurality of lipid particles. Inpreferred embodiments, the active agent or therapeutic agent (e.g.,nucleic acid) is fully encapsulated within the lipid portion of thelipid particles (e.g., SNALP), such that from about 30% to about 100%,from about 40% to about 100%, from about 50% to about 100%, from about60% to about 100%, from about 70% to about 100%, from about 80% to about100%, from about 90% to about 100%, from about 30% to about 95%, fromabout 40% to about 95%, from about 50% to about 95%, from about 60% toabout 95%, %, from about 70% to about 95%, from about 80% to about 95%,from about 85% to about 95%, from about 90% to about 95%, from about 30%to about 90%, from about 40% to about 90%, from about 50% to about 90%,from about 60% to about 90%, from about 70% to about 90%, from about 80%to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%(or any fraction thereof or range therein) of the lipid particles (e.g.,SNALP) have the active agent or therapeutic agent encapsulated therein.

Typically, the lipid particles (e.g., SNALP) of the invention have alipid:active agent (e.g., lipid:nucleic acid) ratio (mass/mass ratio) offrom about 1 to about 100. In some instances, the lipid:active agent(e.g., lipid:nucleic acid) ratio (mass/mass ratio) ranges from about 1to about 50, from about 2 to about 25, from about 3 to about 20, fromabout 4 to about 15, or from about 5 to about 10. In preferredembodiments, the lipid particles of the invention have a lipid:activeagent (e.g., lipid:nucleic acid) ratio (mass/mass ratio) of from about 5to about 15, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (orany fraction thereof or range therein).

Typically, the lipid particles (e.g., SNALP) of the invention have amean diameter of from about 40 nm to about 150 nm. In preferredembodiments, the lipid particles (e.g., SNALP) of the invention have amean diameter of from about 40 nm to about 130 nm, from about 40 nm toabout 120 nm, from about 40 nm to about 100 nm, from about 50 nm toabout 120 nm, from about 50 nm to about 100 nm, from about 60 nm toabout 120 nm, from about 60 nm to about 110 nm, from about 60 nm toabout 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about80 nm, from about 70 nm to about 120 nm, from about 70 nm to about 110nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm,from about 70 nm to about 80 nm, from about 40 nm to about 90 nm, fromabout 45 nm to about 85, or from about 50 nm to abut 80 nm, or less thanabout 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm or 60 nm (or anyfraction thereof or range therein).

In one specific embodiment of the invention, the composition comprises:a plurality of nucleic acid-lipid particles, wherein each particle inthe plurality of particles comprises: (a) one or more unmodified and/ormodified interfering RNA (e.g., siRNA, aiRNA, miRNA) that silence targetgene expression; (b) a cationic lipid comprising from about 56.5 mol %to about 66.5 mol % of the total lipid present in the particle; (c) anon-cationic lipid comprising from about 31.5 mol % to about 42.5 mol %of the total lipid present in the particle; and (d) a conjugated lipidthat inhibits aggregation of particles comprising from about 1 mol % toabout 2 mol % of the total lipid present in the particle, wherein atleast about 95% of the particles in the plurality of particles have anon-lamellar morphology. This specific embodiment of SNALP is generallyreferred to herein as the “1:62” formulation. In a preferred embodiment,the cationic lipid is DLinDMA or DLin-K-C2-DMA (“XTC2”), thenon-cationic lipid is cholesterol, and the conjugated lipid is a PEG-DAAconjugate. Although these are preferred embodiments of the 1:62formulation, those of skill in the art will appreciate that othercationic lipids, non-cationic lipids (including other cholesterolderivatives), and conjugated lipids can be used in the 1:62 formulationas described herein.

In another specific embodiment of the invention, the compositioncomprises: a plurality of nucleic acid-lipid particles, wherein eachparticle in the plurality of particles comprises: (a) one or moreunmodified and/or modified interfering RNA (e.g., siRNA, aiRNA, miRNA)that silence target gene expression; (b) a cationic lipid comprisingfrom about 52 mol % to about 62 mol % of the total lipid present in theparticle; (c) a non-cationic lipid comprising from about 36 mol % toabout 47 mol % of the total lipid present in the particle; and (d) aconjugated lipid that inhibits aggregation of particles comprising fromabout 1 mol % to about 2 mol % of the total lipid present in theparticle, wherein at least about 95% of the particles in the pluralityof particles have a non-lamellar morphology. This specific embodiment ofSNALP is generally referred to herein as the “1:57” formulation. In onepreferred embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA(“XTC2”), the non-cationic lipid is a mixture of a phospholipid (such asDPPC) and cholesterol, wherein the phospholipid comprises from about 5mol % to about 9 mol % of the total lipid present in the particle (e.g.,about 7.1 mol %) and the cholesterol (or cholesterol derivative)comprises from about 32 mol % to about 37 mol % of the total lipidpresent in the particle (e.g., about 34.3 mol %), and the PEG-lipid is aPEG-DAA (e.g., PEG-cDMA). In another preferred embodiment, the cationiclipid is DLinDMA or DLin-K-C2-DMA (“XTC2”), the non-cationic lipid is amixture of a phospholipid (such as DPPC) and cholesterol, wherein thephospholipid comprises from about 15 mol % to about 25 mol % of thetotal lipid present in the particle (e.g., about 20 mol %) and thecholesterol (or cholesterol derivative) comprises from about 15 mol % toabout 25 mol % of the total lipid present in the particle (e.g., about20 mol %), and the PEG-lipid is a PEG-DAA (e.g., PEG-cDMA). Althoughthese are preferred embodiments of the 1:57 formulation, those of skillin the art will appreciate that other cationic lipids, non-cationiclipids (including other phospholipids and other cholesterolderivatives), and conjugated lipids can be used in the 1:57 formulationas described herein.

In preferred embodiments, the 1:62 SNALP formulation is athree-component system which is phospholipid-free and comprises about1.5 mol % PEG-cDMA (or PEG-cDSA), about 61.5 mol % DLinDMA (or XTC2),and about 36.9 mol % cholesterol (or derivative thereof). In otherpreferred embodiments, the 1:57 SNALP formulation is a four-componentsystem which comprises about 1.4 mol % PEG-cDMA (or PEG-cDSA), about57.1 mol % DLinDMA (or XTC2), about 7.1 mol % DPPC, and about 34.3 mol %cholesterol (or derivative thereof). In yet other preferred embodiments,the 1:57 SNALP formulation is a four-component system which comprisesabout 1.4 mol % PEG-cDMA (or PEG-cDSA), about 57.1 mol % DLinDMA (orXTC2), about 20 mol % DPPC, and about 20 mol % cholesterol (orderivative thereof). It should be understood that these SNALPformulations are target formulations, and that the amount of lipid (bothcationic and non-cationic) present and the amount of lipid conjugatepresent in the SNALP formulations may vary.

In yet another specific embodiment of the invention, the compositioncomprises: a plurality of nucleic acid-lipid particles, wherein eachparticle in the plurality of particles comprises: (a) a nucleic acid(e.g., an interfering RNA); (b) a cationic lipid comprising from about50 mol % to about 60 mol % of the total lipid present in the particle;(c) a mixture of a phospholipid and cholesterol or a derivative thereofcomprising from about 35 mol % to about 45 mol % of the total lipidpresent in the particle; and (d) a PEG-lipid conjugate comprising fromabout 5 mol % to about 10 mol % of the total lipid present in theparticle, wherein at least about 95% of the particles in the pluralityof particles have a non-lamellar morphology. This embodiment of nucleicacid-lipid particle is generally referred to herein as the “7:54”formulation.

In still another specific embodiment of the invention, the compositioncomprises: a plurality of nucleic acid-lipid particles, wherein eachparticle in the plurality of particles comprises: (a) a nucleic acid(e.g., an interfering RNA); (b) a cationic lipid comprising from about55 mol % to about 65 mol % of the total lipid present in the particle;(c) cholesterol or a derivative thereof comprising from about 30 mol %to about 40 mol % of the total lipid present in the particle; and (d) aPEG-lipid conjugate comprising from about 5 mol % to about 10 mol % ofthe total lipid present in the particle, wherein at least about 95% ofthe particles in the plurality of particles have a non-lamellarmorphology. This embodiment of nucleic acid-lipid particle is generallyreferred to herein as the “7:58” formulation.

The present invention also provides a pharmaceutical compositioncomprising a lipid particle (e.g., SNALP) described herein and apharmaceutically acceptable carrier.

In a further aspect, the present invention provides a method forintroducing one or more active agents or therapeutic agents (e.g.,nucleic acid) into a cell, comprising contacting the cell with a lipidparticle (e.g., SNALP) described herein. In one embodiment, the cell isin a mammal and the mammal is a human. In another embodiment, thepresent invention provides a method for the in vivo delivery of one ormore active agents or therapeutic agents (e.g., nucleic acid),comprising administering to a mammalian subject a lipid particle (e.g.,SNALP) described herein. In a preferred embodiment, the mode ofadministration includes, but is not limited to, oral, intranasal,intravenous, intraperitoneal, intramuscular, intra-articular,intralesional, intratracheal, subcutaneous, and intradermal. Preferably,the mammalian subject is a human.

In one embodiment, at least about 5%, 10%, 15%, 20%, or 25% of the totalinjected dose of the lipid particles (e.g., SNALP) is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% ofthe total injected dose of the lipid particles (e.g., SNALP) is presentin plasma about 8, 12, 24, 36, or 48 hours after injection. In certaininstances, more than about 10% of a plurality of the particles ispresent in the plasma of a mammal about 1 hour after administration. Incertain other instances, the presence of the lipid particles (e.g.,SNALP) is detectable at least about 1 hour after administration of theparticle. In certain embodiments, the presence of an active agent ortherapeutic agent such as an interfering RNA (e.g., siRNA) is detectablein cells of the lung, liver, tumor, or at a site of inflammation atabout 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration. Inother embodiments, downregulation of expression of a target sequence byan active agent or therapeutic agent such as an interfering RNA (e.g.,siRNA) is detectable at about 8, 12, 24, 36, 48, 60, 72 or 96 hoursafter administration. In yet other embodiments, downregulation ofexpression of a target sequence by an active agent or therapeutic agentsuch as an interfering RNA (e.g., siRNA) occurs preferentially in tumorcells or in cells at a site of inflammation. In further embodiments, thepresence or effect of an active agent or therapeutic agent such as aninterfering RNA (e.g., siRNA) in cells at a site proximal or distal tothe site of administration or in cells of the lung, liver, or a tumor isdetectable at about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10,12, 14, 16, 18, 19, 20, 22, 24, 26, or 28 days after administration. Inadditional embodiments, the lipid particles (e.g., SNALP) of theinvention are administered parenterally or intraperitoneally.

In some embodiments, the lipid particles (e.g., SNALP) of the inventionare particularly useful in methods for the therapeutic delivery of oneor more nucleic acids comprising an interfering RNA sequence (e.g.,siRNA). In particular, it is an object of this invention to provide invitro and in vivo methods for treatment of a disease or disorder in amammal (e.g., a rodent such as a mouse or a primate such as a human,chimpanzee, or monkey) by downregulating or silencing the transcriptionand/or translation of one or more target nucleic acid sequences or genesof interest. As a non-limiting example, the methods of the invention areuseful for in vivo delivery of interfering RNA (e.g., siRNA) to theliver and/or tumor of a mammalian subject. In certain embodiments, thedisease or disorder is associated with expression and/or overexpressionof a gene and expression or overexpression of the gene is reduced by theinterfering RNA (e.g., siRNA). In certain other embodiments, atherapeutically effective amount of the lipid particle (e.g., SNALP) maybe administered to the mammal. In some instances, an interfering RNA(e.g., siRNA) is formulated into a SNALP, and the particles areadministered to patients requiring such treatment. In other instances,cells are removed from a patient, the interfering RNA (e.g., siRNA) isdelivered in vitro (e.g., using a SNALP described herein), and the cellsare reinjected into the patient.

In an additional aspect, the present invention provides lipid particles(e.g., SNALP) comprising asymmetrical interfering RNA (aiRNA) moleculesthat silence the expression of a target gene and methods of using suchparticles to silence target gene expression.

In one embodiment, the aiRNA molecule comprises a double-stranded(duplex) region of about 10 to about 25 (base paired) nucleotides inlength, wherein the aiRNA molecule comprises an antisense strandcomprising 5′ and 3′ overhangs, and wherein the aiRNA molecule iscapable of silencing target gene expression.

In certain instances, the aiRNA molecule comprises a double-stranded(duplex) region of about 12-20, 12-19, 12-18, 13-17, or 14-17 (basepaired) nucleotides in length, more typically 12, 13, 14, 15, 16, 17,18, 19, or 20 (base paired) nucleotides in length. In certain otherinstances, the 5′ and 3′ overhangs on the antisense strand comprisesequences that are complementary to the target RNA sequence, and mayoptionally further comprise nontargeting sequences. In some embodiments,each of the 5′ and 3′ overhangs on the antisense strand comprises orconsists of one, two, three, four, five, six, seven, or morenucleotides.

In other embodiments, the aiRNA molecule comprises modified nucleotidesselected from the group consisting of 2′OMe nucleotides, 2′Fnucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNAnucleotides, and mixtures thereof. In a preferred embodiment, the aiRNAmolecule comprises 2′OMe nucleotides. As a non-limiting example, the2′OMe nucleotides may be selected from the group consisting of2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, and mixturesthereof.

In a related aspect, the present invention provides lipid particles(e.g., SNALP) comprising microRNA (miRNA) molecules that silence theexpression of a target gene and methods of using such compositions tosilence target gene expression.

In one embodiment, the miRNA molecule comprises about 15 to about 60nucleotides in length, wherein the miRNA molecule is capable ofsilencing target gene expression.

In certain instances, the miRNA molecule comprises about 15-50, 15-40,or 15-30 nucleotides in length, more typically about 15-25 or 19-25nucleotides in length, and are preferably about 20-24, 21-22, or 21-23nucleotides in length. In a preferred embodiment, the miRNA molecule isa mature miRNA molecule targeting an RNA sequence of interest.

In some embodiments, the miRNA molecule comprises modified nucleotidesselected from the group consisting of 2′OMe nucleotides, 2′Fnucleotides, 2′-deoxy nucleotides, 2′-O-MOE nucleotides, LNAnucleotides, and mixtures thereof. In a preferred embodiment, the miRNAmolecule comprises 2′OMe nucleotides. As a non-limiting example, the2′OMe nucleotides may be selected from the group consisting of2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, and mixturesthereof.

As such, the lipid particles of the invention (e.g., SNALP) areadvantageous and suitable for use in the administration of active agentsor therapeutic agents such as nucleic acid (e.g., interfering RNA suchas siRNA, aiRNA, and/or miRNA) to a subject (e.g., a mammal such as ahuman) because they are stable in circulation, of a size required forpharmacodynamic behavior resulting in access to extravascular sites, andare capable of reaching target cell populations.

IV. Active Agents

Active agents (e.g., therapeutic agents) include any molecule orcompound capable of exerting a desired effect on a cell, tissue, organ,or subject. Such effects may be, e.g., biological, physiological, and/orcosmetic. Active agents may be any type of molecule or compoundincluding, but not limited to, nucleic acids, peptides, polypeptides,small molecules, and mixtures thereof. Non-limiting examples of nucleicacids include interfering RNA molecules (e.g., dsRNA such as siRNA,Dicer-substrate dsRNA, shRNA, aiRNA, and/or miRNA), antisenseoligonucleotides, plasmids, ribozymes, immunostimulatoryoligonucleotides, and mixtures thereof. Examples of peptides orpolypeptides include, without limitation, antibodies (e.g., polyclonalantibodies, monoclonal antibodies, antibody fragments; humanizedantibodies, recombinant antibodies, recombinant human antibodies, and/orPrimatized™ antibodies), cytokines, growth factors, apoptotic factors,differentiation-inducing factors, cell-surface receptors and theirligands, hormones, and mixtures thereof. Examples of small moleculesinclude, but are not limited to, small organic molecules or compoundssuch as any conventional agent or drug known to those of skill in theart.

In some embodiments, the active agent is a therapeutic agent, or a saltor derivative thereof. Therapeutic agent derivatives may betherapeutically active themselves or they may be prodrugs, which becomeactive upon further modification. Thus, in one embodiment, a therapeuticagent derivative retains some or all of the therapeutic activity ascompared to the unmodified agent, while in another embodiment, atherapeutic agent derivative is a prodrug that lacks therapeuticactivity, but becomes active upon further modification.

A. Nucleic Acids

In certain embodiments, lipid particles of the present invention areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle (e.g., SNALP). In some embodiments, the nucleic acid is fullyencapsulated in the lipid particle. As used herein, the term “nucleicacid” includes any oligonucleotide or polynucleotide, with fragmentscontaining up to 60 nucleotides generally termed oligonucleotides, andlonger fragments termed polynucleotides. In particular embodiments,oligonucletoides of the invention are from about 15 to about 60nucleotides in length. Nucleic acid may be administered alone in thelipid particles of the invention, or in combination (e.g.,co-administered) with lipid particles of the invention comprisingpeptides, polypeptides, or small molecules such as conventional drugs.Similarly, when used to treat a cell proliferative disorder such ascancer, the nucleic acid, such as the interfering RNA molecule (e.g.,siRNA), can be administered alone or co-administered (i.e., concurrentlyor consecutively) with conventional agents used to treat, e.g., a cellproliferative disorder such as cancer. Such agents include chemotherapydrugs as well as conventional hormonal, immunotherapeutic, and/orradiotherapeutic agents.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally-occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also include polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake, reduced immunogenicity, and increasedstability in the presence of nucleases.

Oligonucleotides are generally classified as deoxyribooligonucleotidesor ribooligonucleotides. A deoxyribooligonucleotide consists of a5-carbon sugar called deoxyribose joined covalently to phosphate at the5′ and 3′ carbons of this sugar to form an alternating, unbranchedpolymer. A ribooligonucleotide consists of a similar repeating structurewhere the 5-carbon sugar is ribose.

The nucleic acid that is present in a nucleic acid-lipid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. In preferredembodiments, the nucleic acids are double-stranded RNA. Examples ofdouble-stranded RNA are described herein and include, e.g., siRNA andother RNAi agents such as Dicer-substrate dsRNA, shRNA, aiRNA, andpre-miRNA. In other preferred embodiments, the nucleic acids aresingle-stranded nucleic acids. Single-stranded nucleic acids include,e.g., antisense oligonucleotides, ribozymes, mature miRNA, andtriplex-forming oligonucleotides. In further embodiments, the nucleicacids are double-stranded DNA. Examples of double-stranded DNA include,e.g., DNA-DNA hybrids comprising a DNA sense strand and a DNA antisensestrand as described in PCT Publicaiton No. WO 2004/104199, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

Nucleic acids of the invention may be of various lengths, generallydependent upon the particular form of nucleic acid. For example, inparticular embodiments, plasmids or genes may be from about 1,000 toabout 100,000 nucleotide residues in length. In particular embodiments,oligonucleotides may range from about 10 to about 100 nucleotides inlength. In various related embodiments, oligonucleotides, bothsingle-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 60 nucleotides, from about 15 to about 60nucleotides, from about 20 to about 50 nucleotides, from about 15 toabout 30 nucleotides, or from about 20 to about 30 nucleotides inlength.

In particular embodiments, an oligonucleotide (or a strand thereof) ofthe invention specifically hybridizes to or is complementary to a targetpolynucleotide sequence. The terms “specifically hybridizable” and“complementary” as used herein indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. In preferred embodiments,an oligonucleotide is specifically hybridizable when binding of theoligonucleotide to the target sequence interferes with the normalfunction of the target sequence to cause a loss of utility or expressionthere from, and there is a sufficient degree of complementarity to avoidnon-specific binding of the oligonucleotide to non-target sequencesunder conditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, or, in the case of in vitro assays, under conditions in whichthe assays are conducted. Thus, the oligonucleotide may include 1, 2, 3,or more base substitutions as compared to the region of a gene or mRNAsequence that it is targeting or to which it specifically hybridizes.

1. siRNA

The siRNA component of the nucleic acid-lipid particles of the presentinvention is capable of silencing the expression of a target gene ofinterest, such as PLK-1. Each strand of the siRNA duplex is typicallyabout 15 to about 60 nucleotides in length, preferably about 15 to about30 nucleotides in length. In certain embodiments, the siRNA comprises atleast one modified nucleotide. The modified siRNA is generally lessimmunostimulatory than a corresponding unmodified siRNA sequence andretains RNAi activity against the target gene of interest. In someembodiments, the modified siRNA contains at least one 2′OMe purine orpyrimidine nucleotide such as a 2′OMe-guanosine, 2′OMe-uridine,2′OMe-adenosine, and/or 2′OMe-cytosine nucleotide. The modifiednucleotides can be present in one strand (i.e., sense or antisense) orboth strands of the siRNA. In some preferred embodiments, one or more ofthe uridine and/or guanosine nucleotides are modified (e.g.,2′OMe-modified) in one strand (i.e., sense or antisense) or both strandsof the siRNA. In these embodiments, the modified siRNA can furthercomprise one or more modified (e.g., 2′OMe-modified) adenosine and/ormodified (e.g., 2′OMe-modified) cytosine nucleotides. In other preferredembodiments, only uridine and/or guanosine nucleotides are modified(e.g., 2′OMe-modified) in one strand (i.e., sense or antisense) or bothstrands of the siRNA. The siRNA sequences may have overhangs (e.g., 3′or 5′ overhangs as described in Elbashir et al., Genes Dev., 15:188(2001) or Nykänen et al., Cell, 107:309 (2001)), or may lack overhangs(i.e., have blunt ends).

In particular embodiments, the selective incorporation of modifiednucleotides such as 2′OMe uridine and/or guanosine nucleotides into thedouble-stranded region of either or both strands of the siRNA reduces orcompletely abrogates the immune response to that siRNA molecule. Incertain instances, the immunostimulatory properties of specific siRNAsequences and their ability to silence gene expression can be balancedor optimized by the introduction of minimal and selective 2′OMemodifications within the double-stranded region of the siRNA duplex.This can be achieved at therapeutically viable siRNA doses withoutcytokine induction, toxicity, and off-target effects associated with theuse of unmodified siRNA.

The modified siRNA generally comprises from about 1% to about 100%(e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%,28%, 29%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100%) modified nucleotides in the double-stranded region ofthe siRNA duplex. In certain embodiments, one, two, three, four, five,six, seven, eight, nine, ten, or more of the nucleotides in thedouble-stranded region of the siRNA comprise modified nucleotides. Incertain other embodiments, some or all of the modified nucleotides inthe double-stranded region of the siRNA are 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more nucleotides apart from each other. In one preferredembodiment, none of the modified nucleotides in the double-strandedregion of the siRNA are adjacent to each other (e.g., there is a gap ofat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified nucleotides betweeneach modified nucleotide). In another preferred embodiment, at least twoof the modified nucleotides in the double-stranded region of the siRNAare adjacent to each other (e.g., there are no unmodified nucleotidesbetween two or more modified nucleotides). In other preferredembodiments, at least three, at least four, or at least five of themodified nucleotides in the double-stranded region of the siRNA areadjacent to each other.

In some embodiments, less than about 50% (e.g., less than about 49%,48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, or 36%,preferably less than about 35%, 34%, 33%, 32%, 31%, or 30%) of thenucleotides in the double-stranded region of the siRNA comprise modified(e.g., 2′OMe) nucleotides. In one aspect of these embodiments, less thanabout 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, lessthan about 50% of the nucleotides in the double-stranded region of thesiRNA comprise 2′OMe nucleotides, wherein the siRNA comprises 2′OMenucleotides in both strands of the siRNA, wherein the siRNA comprises atleast one 2′OMe-guanosine nucleotide and at least one 2′OMe-uridinenucleotide, and wherein the siRNA does not comprise 2′OMe-cytosinenucleotides in the double-stranded region. In another aspect of theseembodiments, less than about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the 2′OMe nucleotides in the double-stranded region are notadjacent to each other.

In other embodiments, from about 1% to about 50% (e.g., from about5%-50%, 10%-50%, 15%-50%, 20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%,45%-50%, 5%-45%, 10%-45%, 15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%,40%-45%, 5%-40%, 10%-40%, 15%-40%, 20%-40%, 25%-40%, 25%-39%, 25%-38%,25%-37%, 25%-36%, 26%-39%, 26%-38%, 26%-37%, 26%-36%, 27%-39%, 27%-38%,27%-37%, 27%-36%, 28%-39%, 28%-38%, 28%-37%, 28%-36%, 29%-39%, 29%-38%,29%-37%, 29%-36%, 30%-40%, 30%-39%, 30%-38%, 30%-37%, 30%-36%, 31%-39%,31%-38%, 31%-37%, 31%-36%, 32%-39%, 32%-38%, 32%-37%, 32%-36%, 33%-39%,33%-38%, 33%-37%, 33%-36%, 34%-39%, 34%-38%, 34%-37%, 34%-36%, 35%-40%,5%-35%, 10%-35%, 15%-35%, 20%-35%, 21%-35%, 22%-35%, 23%-35%, 24%-35%,25%-35%, 26%-35%, 27%-35%, 28%-35%, 29%-35%, 30%-35%, 31%-35%, 32%-35%,33%-35%, 34%-35%, 30%-34%, 31%-34%, 32%-34%, 33%-34%, 30%-33%, 31%-33%,32%-33%, 30%-32%, 31%-32%, 25%-34%, 25%-33%, 25%-32%, 25%-31%, 26%-34%,26%-33%, 26%-32%, 26%-31%, 27%-34%, 27%-33%, 27%-32%, 27%-31%, 28%-34%,28%-33%, 28%-32%, 28%-31%, 29%-34%, 29%-33%, 29%-32%, 29%-31%, 5%-30%,10%-30%, 15%-30%, 20%-34%, 20%-33%, 20%-32%, 20%-31%, 20%-30%, 21%-30%,22%-30%, 23%-30%, 24%-30%, 25%-30%, 25%-29%, 25%-28%, 25%-27%, 25%-26%,26%-30%, 26%-29%, 26%-28%, 26%-27%, 27%-30%, 27%-29%, 27%-28%, 28%-30%,28%-29%, 29%-30%, 5%-25%, 10%-25%, 15%-25%, 20%-29%, 20%-28%, 20%-27%,20%-26%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%, 10%-15%, or 5%-10%)of the nucleotides in the double-stranded region of the siRNA comprisemodified nucleotides. In one aspect of these embodiments, from about 1%to about 50% of the uridine and/or guanosine nucleotides in thedouble-stranded region of one or both strands of the siRNA areselectively (e.g., only) modified. In another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the siRNA,wherein the siRNA comprises at least one 2′OMe-guanosine nucleotide andat least one 2′OMe-uridine nucleotide, and wherein 2′OMe-guanosinenucleotides and 2′OMe-uridine nucleotides are the only 2′OMe nucleotidespresent in the double-stranded region. In yet another aspect of theseembodiments, from about 1% to about 50% of the nucleotides in thedouble-stranded region of the siRNA comprise 2′OMe nucleotides, whereinthe siRNA comprises 2′OMe nucleotides in both strands of the modifiedsiRNA, wherein the siRNA comprises 2′OMe nucleotides selected from thegroup consisting of 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, and mixtures thereof, andwherein the siRNA does not comprise 2′OMe-cytosine nucleotides in thedouble-stranded region. In a further aspect of these embodiments, fromabout 1% to about 50% of the nucleotides in the double-stranded regionof the siRNA comprise 2′OMe nucleotides, wherein the siRNA comprises2′OMe nucleotides in both strands of the siRNA, wherein the siRNAcomprises at least one 2′OMe-guanosine nucleotide and at least one2′OMe-uridine nucleotide, and wherein the siRNA does not comprise2′OMe-cytosine nucleotides in the double-stranded region. In anotheraspect of these embodiments, from about 1% to about 50% of thenucleotides in the double-stranded region of the siRNA comprise 2′OMenucleotides, wherein the siRNA comprises 2′OMe nucleotides in bothstrands of the modified siRNA, wherein the siRNA comprises 2′OMenucleotides selected from the group consisting of 2′OMe-guanosinenucleotides, 2′OMe-uridine nucleotides, 2′OMe-adenosine nucleotides, andmixtures thereof, and wherein the 2′OMe nucleotides in thedouble-stranded region are not adjacent to each other.

In certain embodiments, the siRNA component of the nucleic acid-lipidparticles of the present invention (e.g., SNALP) comprises an asymmetricsiRNA duplex as described in PCT Publication No. WO 2004/078941, whichcomprises a double-stranded region consisting of a DNA sense strand andan RNA antisense strand (e.g., a DNA-RNA hybrid), wherein a blockingagent is located on the siRNA duplex. In some instances, the asymmetricsiRNA duplex can be chemically modified as described herein. Othernon-limiting examples of asymmetric siRNA duplexes are described in PCTPublication No. WO 2006/074108, which discloses self-protectedoligonucleotides comprising a region having a sequence complementary toone, two, three, or more same or different target mRNA sequences (e.g.,multivalent siRNAs) and one or more self-complementary regions. Yetother non-limiting examples of asymmetric siRNA duplexes are describedin PCT Publication No. WO 2009/076321, which discloses self-formingasymmetric precursor polynucleotides comprising a targeting regioncomprising a polynucleotide sequence complementary to a region of one,two, three, or more same or different target mRNA sequences (e.g.,multivalent siRNAs); a first self-complementary region; and a secondself-complementary region, wherein the first and secondself-complementary regions are located one at each end of the targetingregion and both self-complementary regions form stem-loop structures,wherein the first self-complementary region is capable of being cleavedby a RNase III endoribonuclease that is not a class IV DICERendoribonuclease, and wherein both self-complementary regions comprise anucleotide sequence that is complementary to a region of the target genesequence, but wherein a portion of the target sequence present in thetargeting region does not have a complementary sequence in either of theself-complementary regions. The disclosures of each of the above patentdocuments are herein incorporated by reference in their entirety for allpurposes.

Additional ranges, percentages, and patterns of modifications that maybe introduced into siRNA are described in U.S. Patent Publication No.20070135372, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

a) Selection of siRNA Sequences

Suitable siRNA sequences can be identified using any means known in theart. Typically, the methods described in Elbashir et al., Nature,411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) arecombined with rational design rules set forth in Reynolds et al., NatureBiotech., 22(3):326-330 (2004).

As a non-limiting example, the nucleotide sequence 3′ of the AUG startcodon of a transcript from the target gene of interest may be scannedfor dinucleotide sequences (e.g., AA, NA, CC, GG, or UU, wherein N═C, G,or U) (see, e.g., Elbashir et al., EMBO J., 20:6877-6888 (2001)). Thenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences (i.e., a target sequence or a sense strandsequence). Typically, the 19, 21, 23, 25, 27, 29, 31, 33, 35, or morenucleotides immediately 3′ to the dinucleotide sequences are identifiedas potential siRNA sequences. In some embodiments, the dinucleotidesequence is an AA or NA sequence and the 19 nucleotides immediately 3′to the AA or NA dinucleotide are identified as potential siRNAsequences. siRNA sequences are usually spaced at different positionsalong the length of the target gene. To further enhance silencingefficiency of the siRNA sequences, potential siRNA sequences may beanalyzed to identify sites that do not contain regions of homology toother coding sequences, e.g., in the target cell or organism. Forexample, a suitable siRNA sequence of about 21 base pairs typically willnot have more than 16-17 contiguous base pairs of homology to codingsequences in the target cell or organism. If the siRNA sequences are tobe expressed from an RNA Pol III promoter, siRNA sequences lacking morethan 4 contiguous A's or T's are selected.

Once a potential siRNA sequence has been identified, a complementarysequence (i.e., an antisense strand sequence) can be designed. Apotential siRNA sequence can also be analyzed using a variety ofcriteria known in the art. For example, to enhance their silencingefficiency, the siRNA sequences may be analyzed by a rational designalgorithm to identify sequences that have one or more of the followingfeatures: (1) G/C content of about 25% to about 60% G/C; (2) at least 3A/Us at positions 15-19 of the sense strand; (3) no internal repeats;(4) an A at position 19 of the sense strand; (5) an A at position 3 ofthe sense strand; (6) a U at position 10 of the sense strand; (7) no G/Cat position 19 of the sense strand; and (8) no G at position 13 of thesense strand. siRNA design tools that incorporate algorithms that assignsuitable values of each of these features and are useful for selectionof siRNA can be found at, e.g.,http://ihome.ust.hk/˜bokcmho/siRNA/siRNA.html. One of skill in the artwill appreciate that sequences with one or more of the foregoingcharacteristics may be selected for further analysis and testing aspotential siRNA sequences.

Additionally, potential siRNA sequences with one or more of thefollowing criteria can often be eliminated as siRNA: (1) sequencescomprising a stretch of 4 or more of the same base in a row; (2)sequences comprising homopolymers of Gs (i.e., to reduce possiblenon-specific effects due to structural characteristics of thesepolymers; (3) sequences comprising triple base motifs (e.g., GGG, CCC,AAA, or TTT); (4) sequences comprising stretches of 7 or more G/Cs in arow; and (5) sequences comprising direct repeats of 4 or more baseswithin the candidates resulting in internal fold-back structures.However, one of skill in the art will appreciate that sequences with oneor more of the foregoing characteristics may still be selected forfurther analysis and testing as potential siRNA sequences.

In some embodiments, potential siRNA sequences may be further analyzedbased on siRNA duplex asymmetry as described in, e.g., Khvorova et al.,Cell, 115:209-216 (2003); and Schwarz et al., Cell, 115:199-208 (2003).In other embodiments, potential siRNA sequences may be further analyzedbased on secondary structure at the target site as described in, e.g.,Luo et al., Biophys. Res. Commun., 318:303-310 (2004). For example,secondary structure at the target site can be modeled using the Mfoldalgorithm (available at http://mfold.burnet.edu.au/rna_form) to selectsiRNA sequences which favor accessibility at the target site where lesssecondary structure in the form of base-pairing and stem-loops ispresent.

Once a potential siRNA sequence has been identified, the sequence can beanalyzed for the presence of any immunostimulatory properties, e.g.,using an in vitro cytokine assay or an in vivo animal model. Motifs inthe sense and/or antisense strand of the siRNA sequence such as GU-richmotifs (e.g., 5′-GU-3′, 5′-UGU-3′, 5′-GUGU-3′, 5′-UGUGU-3′, etc.) canalso provide an indication of whether the sequence may beimmunostimulatory. Once an siRNA molecule is found to beimmunostimulatory, it can then be modified to decrease itsimmunostimulatory properties as described herein. As a non-limitingexample, an siRNA sequence can be contacted with a mammalian respondercell under conditions such that the cell produces a detectable immuneresponse to determine whether the siRNA is an immunostimulatory or anon-immunostimulatory siRNA. The mammalian responder cell may be from anaïve mammal (i.e., a mammal that has not previously been in contactwith the gene product of the siRNA sequence). The mammalian respondercell may be, e.g., a peripheral blood mononuclear cell (PBMC), amacrophage, and the like. The detectable immune response may compriseproduction of a cytokine or growth factor such as, e.g., TNF-α, IFN-α,IFN-0, IFN-γ, IL-6, IL-8, IL-12, or a combination thereof. An siRNAidentified as being immunostimulatory can then be modified to decreaseits immunostimulatory properties by replacing at least one of thenucleotides on the sense and/or antisense strand with modifiednucleotides. For example, less than about 30% (e.g., less than about30%, 25%, 20%, 15%, 10%, or 5%) of the nucleotides in thedouble-stranded region of the siRNA duplex can be replaced with modifiednucleotides such as 2′OMe nucleotides. The modified siRNA can then becontacted with a mammalian responder cell as described above to confirmthat its immunostimulatory properties have been reduced or abrogated.

Suitable in vitro assays for detecting an immune response include, butare not limited to, the double monoclonal antibody sandwich immunoassaytechnique of David et al. (U.S. Pat. No. 4,376,110);monoclonal-polyclonal antibody sandwich assays (Wide et al., in Kirkhamand Hunter, eds., Radioimmunoassay Methods, E. and S. Livingstone,Edinburgh (1970)); the “Western blot” method of Gordon et al. (U.S. Pat.No. 4,452,901); immunoprecipitation of labeled ligand (Brown et al., J.Biol. Chem., 255:4980-4983 (1980)); enzyme-linked immunosorbent assays(ELISA) as described, for example, by Raines et al., J. Biol. Chem.,257:5154-5160 (1982); immunocytochemical techniques, including the useof fluorochromes (Brooks et al., Clin. Exp. Immunol., 39:477 (1980));and neutralization of activity (Bowen-Pope et al., Proc. Natl. Acad.Sci. USA, 81:2396-2400 (1984)). In addition to the immunoassaysdescribed above, a number of other immunoassays are available, includingthose described in U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876. Thedisclosures of these references are herein incorporated by reference intheir entirety for all purposes.

A non-limiting example of an in vivo model for detecting an immuneresponse includes an in vivo mouse cytokine induction assay as describedin, e.g., Judge et al., Mol. Ther., 13:494-505 (2006). In certainembodiments, the assay that can be performed as follows: (1) siRNA canbe administered by standard intravenous injection in the lateral tailvein; (2) blood can be collected by cardiac puncture about 6 hours afteradministration and processed as plasma for cytokine analysis; and (3)cytokines can be quantified using sandwich ELISA kits according to themanufacturer's instructions (e.g., mouse and human IFN-α (PBLBiomedical; Piscataway, N.J.); human IL-6 and TNF-α (eBioscience; SanDiego, Calif.); and mouse IL-6, TNF-α, and IFN-γ (BD Biosciences; SanDiego, Calif.)).

Monoclonal antibodies that specifically bind cytokines and growthfactors are commercially available from multiple sources and can begenerated using methods known in the art (see, e.g., Kohler et al.,Nature, 256: 495-497 (1975) and Harlow and Lane, ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publication, New York (1999)).Generation of monoclonal antibodies has been previously described andcan be accomplished by any means known in the art (Buhring et al., inHybridoma, Vol. 10, No. 1, pp. 77-78 (1991)). In some methods, themonoclonal antibody is labeled (e.g., with any composition detectable byspectroscopic, photochemical, biochemical, electrical, optical, orchemical means) to facilitate detection.

b) Generating siRNA Molecules

siRNA can be provided in several forms including, e.g., as one or moreisolated small-interfering RNA (siRNA) duplexes, as longerdouble-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from atranscriptional cassette in a DNA plasmid. In some embodiments, siRNAmay be produced enzymatically or by partial/total organic synthesis, andmodified ribonucleotides can be introduced by in vitro enzymatic ororganic synthesis. In certain instances, each strand is preparedchemically. Methods of synthesizing RNA molecules are known in the art,e.g., the chemical synthesis methods as described in Verma and Eckstein(1998) or as described herein.

An RNA population can be used to provide long precursor RNAs, or longprecursor RNAs that have substantial or complete identity to a selectedtarget sequence can be used to make the siRNA. The RNAs can be isolatedfrom cells or tissue, synthesized, and/or cloned according to methodswell known to those of skill in the art. The RNA can be a mixedpopulation (obtained from cells or tissue, transcribed from cDNA,subtracted, selected, etc.), or can represent a single target sequence.RNA can be naturally occurring (e.g., isolated from tissue or cellsamples), synthesized in vitro (e.g., using T7 or SP6 polymerase and PCRproducts or a cloned cDNA), or chemically synthesized.

To form a long dsRNA, for synthetic RNAs, the complement is alsotranscribed in vitro and hybridized to form a dsRNA. If a naturallyoccuring RNA population is used, the RNA complements are also provided(e.g., to form dsRNA for digestion by E. coli RNAse III or Dicer), e.g.,by transcribing cDNAs corresponding to the RNA population, or by usingRNA polymerases. The precursor RNAs are then hybridized to form doublestranded RNAs for digestion. The dsRNAs can be directly administered toa subject or can be digested in vitro prior to administration.

Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids,making and screening cDNA libraries, and performing PCR are well knownin the art (see, e.g., Gubler and Hoffman, Gene, 25:263-269 (1983);Sambrook et al., supra; Ausubel et al., supra), as are PCR methods (see,U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide toMethods and Applications (Innis et al., eds, 1990)). Expressionlibraries are also well known to those of skill in the art. Additionalbasic texts disclosing the general methods of use in this inventioninclude Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed.1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Current Protocols in Molecular Biology (Ausubel et al.,eds., 1994). The disclosures of these references are herein incorporatedby reference in their entirety for all purposes.

Preferably, siRNA are chemically synthesized. The oligonucleotides thatcomprise the siRNA molecules of the invention can be synthesized usingany of a variety of techniques known in the art, such as those describedin Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al.,Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res.,23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59(1997). The synthesis of oligonucleotides makes use of common nucleicacid protecting and coupling groups, such as dimethoxytrityl at the5′-end and phosphoramidites at the 3′-end. As a non-limiting example,small scale syntheses can be conducted on an Applied Biosystemssynthesizer using a 0.2 μmol scale protocol. Alternatively, syntheses atthe 0.2 μmol scale can be performed on a 96-well plate synthesizer fromProtogene (Palo Alto, Calif.). However, a larger or smaller scale ofsynthesis is also within the scope of this invention. Suitable reagentsfor oligonucleotide synthesis, methods for RNA deprotection, and methodsfor RNA purification are known to those of skill in the art.

siRNA molecules can also be synthesized via a tandem synthesistechnique, wherein both strands are synthesized as a single continuousoligonucleotide fragment or strand separated by a cleavable linker thatis subsequently cleaved to provide separate fragments or strands thathybridize to form the siRNA duplex. The linker can be a polynucleotidelinker or a non-nucleotide linker. The tandem synthesis of siRNA can bereadily adapted to both multiwell/multiplate synthesis platforms as wellas large scale synthesis platforms employing batch reactors, synthesiscolumns, and the like. Alternatively, siRNA molecules can be assembledfrom two distinct oligonucleotides, wherein one oligonucleotidecomprises the sense strand and the other comprises the antisense strandof the siRNA. For example, each strand can be synthesized separately andjoined together by hybridization or ligation following synthesis and/ordeprotection. In certain other instances, siRNA molecules can besynthesized as a single continuous oligonucleotide fragment, where theself-complementary sense and antisense regions hybridize to form ansiRNA duplex having hairpin secondary structure.

c) Modifying siRNA Sequences

In certain aspects, siRNA molecules comprise a duplex having two strandsand at least one modified nucleotide in the double-stranded region,wherein each strand is about 15 to about 60 nucleotides in length.Advantageously, the modified siRNA is less immunostimulatory than acorresponding unmodified siRNA sequence, but retains the capability ofsilencing the expression of a target sequence. In preferred embodiments,the degree of chemical modifications introduced into the siRNA strikes abalance between reduction or abrogation of the immunostimulatoryproperties of the siRNA and retention of RNAi activity. As anon-limiting example, an siRNA molecule that targets a gene of interestcan be minimally modified (e.g., less than about 30%, 25%, 20%, 15%,10%, or 5% modified) at selective uridine and/or guanosine nucleotideswithin the siRNA duplex to eliminate the immune response generated bythe siRNA while retaining its capability to silence target geneexpression.

Examples of modified nucleotides suitable for use in the inventioninclude, but are not limited to, ribonucleotides having a 2′-O-methyl(2′OMe), 2′-deoxy-2′-fluoro (2′F), 2′-deoxy, 5-C-methyl,2′-O-(2-methoxyethyl) (MOE), 4′-thio, 2′-amino, or 2′-C-allyl group.Modified nucleotides having a Northern conformation such as thosedescribed in, e.g., Saenger, Principles of Nucleic Acid Structure,Springer-Verlag Ed. (1984), are also suitable for use in siRNAmolecules. Such modified nucleotides include, without limitation, lockednucleic acid (LNA) nucleotides (e.g., 2′-0,4′-C-methylene-(D-ribofuranosyl) nucleotides), 2′-O-(2-methoxyethyl)(MOE) nucleotides, 2′-methyl-thio-ethyl nucleotides, 2′-deoxy-2′-fluoro(2′F) nucleotides, 2′-deoxy-2′-chloro (2′Cl) nucleotides, and 2′-azidonucleotides. In certain instances, the siRNA molecules described hereininclude one or more G-clamp nucleotides. A G-clamp nucleotide refers toa modified cytosine analog wherein the modifications confer the abilityto hydrogen bond both Watson-Crick and Hoogsteen faces of acomplementary guanine nucleotide within a duplex (see, e.g., Lin et al.,J. Am. Chem. Soc., 120:8531-8532 (1998)). In addition, nucleotideshaving a nucleotide base analog such as, for example, C-phenyl,C-naphthyl, other aromatic derivatives, inosine, azole carboxamides, andnitroazole derivatives such as 3-nitropyrrole, 4-nitroindole,5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res.,29:2437-2447 (2001)) can be incorporated into siRNA molecules.

In certain embodiments, siRNA molecules may further comprise one or morechemical modifications such as terminal cap moieties, phosphate backbonemodifications, and the like. Examples of terminal cap moieties include,without limitation, inverted deoxy abasic residues, glycerylmodifications, 4′,5′-methylene nucleotides, 1-O-D-erythrofuranosyl)nucleotides, 4′-thio nucleotides, carbocyclic nucleotides,1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modifiedbase nucleotides, threo-pentofuranosyl nucleotides, acyclic 3′,4′-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic3,5-dihydroxypentyl nucleotides, 3′-3′-inverted nucleotide moieties,3′-3′-inverted abasic moieties, 3′-2′-inverted nucleotide moieties,3′-2′-inverted abasic moieties, 5′-5′-inverted nucleotide moieties,5′-5′-inverted abasic moieties, 3′-5′-inverted deoxy abasic moieties,5′-amino-alkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropylphosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate,hydroxypropyl phosphate, 1,4-butanediol phosphate, 3′-phosphoramidate,5′-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3′-phosphate,5′-amino, 3′-phosphorothioate, 5′-phosphorothioate, phosphorodithioate,and bridging or non-bridging methylphosphonate or 5′-mercapto moieties(see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron49:1925 (1993)). Non-limiting examples of phosphate backbonemodifications (i.e., resulting in modified internucleotide linkages)include phosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate, carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and alkylsilyl substitutions (see, e.g., Hunziker etal., Nucleic Acid Analogues: Synthesis and Properties, in ModernSynthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39 (1994)). Such chemical modifications canoccur at the 5′-end and/or 3′-end of the sense strand, antisense strand,or both strands of the siRNA. The disclosures of these references areherein incorporated by reference in their entirety for all purposes.

In some embodiments, the sense and/or antisense strand of the siRNAmolecule can further comprise a 3′-terminal overhang having about 1 toabout 4 (e.g., 1, 2, 3, or 4) 2′-deoxy ribonucleotides, modified (e.g.,2′OMe) and/or unmodified uridine ribonucleotides, and/or any othercombination of modified (e.g., 2′OMe) and unmodified nucleotides.

Additional examples of modified nucleotides and types of chemicalmodifications that can be introduced into siRNA molecules are described,e.g., in UK Patent No. GB 2,397,818 B and U.S. Patent Publication Nos.20040192626, 20050282188, and 20070135372, the disclosures of which areherein incorporated by reference in their entirety for all purposes.

The siRNA molecules described herein can optionally comprise one or morenon-nucleotides in one or both strands of the siRNA. As used herein, theterm “non-nucleotide” refers to any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including sugar and/or phosphate substitutions, andallows the remaining bases to exhibit their activity. The group orcompound is abasic in that it does not contain a commonly recognizednucleotide base such as adenosine, guanine, cytosine, uracil, or thymineand therefore lacks a base at the 1′-position.

In other embodiments, chemical modification of the siRNA comprisesattaching a conjugate to the siRNA molecule. The conjugate can beattached at the 5′ and/or 3′-end of the sense and/or antisense strand ofthe siRNA via a covalent attachment such as, e.g., a biodegradablelinker. The conjugate can also be attached to the siRNA, e.g., through acarbamate group or other linking group (see, e.g., U.S. PatentPublication Nos. 20050074771, 20050043219, and 20050158727). In certaininstances, the conjugate is a molecule that facilitates the delivery ofthe siRNA into a cell. Examples of conjugate molecules suitable forattachment to siRNA include, without limitation, steroids such ascholesterol, glycols such as polyethylene glycol (PEG), human serumalbumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates(e.g., folic acid, folate analogs and derivatives thereof), sugars(e.g., galactose, galactosamine, N-acetyl galactosamine, glucose,mannose, fructose, fucose, etc.), phospholipids, peptides, ligands forcellular receptors capable of mediating cellular uptake, andcombinations thereof (see, e.g., U.S. Patent Publication Nos.20030130186, 20040110296, and 20040249178; U.S. Pat. No. 6,753,423).Other examples include the lipophilic moiety, vitamin, polymer, peptide,protein, nucleic acid, small molecule, oligosaccharide, carbohydratecluster, intercalator, minor groove binder, cleaving agent, andcross-linking agent conjugate molecules described in U.S. PatentPublication Nos. 20050119470 and 20050107325. Yet other examples includethe 2′-O-alkyl amine, 2′-O-alkoxyalkyl amine, polyamine, C5-cationicmodified pyrimidine, cationic peptide, guanidinium group, amidininiumgroup, cationic amino acid conjugate molecules described in U.S. PatentPublication No. 20050153337. Additional examples include the hydrophobicgroup, membrane active compound, cell penetrating compound, celltargeting signal, interaction modifier, and steric stabilizer conjugatemolecules described in U.S. Patent Publication No. 20040167090. Furtherexamples include the conjugate molecules described in U.S. PatentPublication No. 20050239739. The type of conjugate used and the extentof conjugation to the siRNA molecule can be evaluated for improvedpharmacokinetic profiles, bioavailability, and/or stability of the siRNAwhile retaining RNAi activity. As such, one skilled in the art canscreen siRNA molecules having various conjugates attached thereto toidentify ones having improved properties and full RNAi activity usingany of a variety of well-known in vitro cell culture or in vivo animalmodels. The disclosures of the above-described patent documents areherein incorporated by reference in their entirety for all purposes.

d) Target Genes

The siRNA component of the nucleic acid-lipid particles of the presentinvention (e.g., SNALP) can be used to downregulate or silence thetranslation (i.e., expression) of a gene of interest. As previouslymentioned, it has been unexpectedly found that the nucleic acid-lipidparticles of the present invention (i.e., SNALP formulations) containingat least one siRNA as disclosed herein show increased potency (i.e.,increased silencing) and/or increased tolerability (e.g., decreasedtoxicity) when targeting a gene of interest in a tumor cell, whencompared to other nucleic acid-lipid particle compositions previouslydescribed. In preferred embodiments, the siRNA silences the expressionof a gene associated with cell proliferation, tumorigenesis, and/or celltransformation (e.g., a cell proliferative disorder such as cancer).Other genes of interest include, but are not limited to, angiogenicgenes, receptor ligand genes, immunomodulator genes (e.g., thoseassociated with inflammatory and autoimmune responses), genes associatedwith metabolic diseases and disorders (e.g., liver diseases anddisorders), genes associated with viral infection and survival, andgenes associated with neurodegenerative disorders.

Genes associated with tumorigenesis or cell transformation (e.g., canceror other neoplasia) include, for example, genes involved in p53ubiquitination, c-Jun ubiquitination, histone deacetylation, cell cycleregulation, transcriptional regulation, and combinations thereof.Non-limiting examples of gene sequences associated with tumorigenesis orcell transformation include serine/threonine kinases such as polo-likekinase 1 (PLK-1) (Genbank Accession No. NM_005030; Barr et al., Nat.Rev. Mol. Cell Biol., 5:429-440 (2004)) and cyclin-dependent kinase 4(CDK4) (Genbank Accession No. NM_000075); ubiquitin ligases such as COP1(RFWD2; Genbank Accession Nos. NM_022457 and NM_001001740) and ring-box1 (RBX1) (ROC1; Genbank Accession No. NM_014248);

tyrosine kinases such as WEE1 (Genbank Accession Nos. NM_003390 andNM_001143976); mitotic kinesins such as Eg5 (KSP, KIF11; GenbankAccession No. NM_004523); transcription factors such as forkhead box M1(FOXM1) (Genbank Accession Nos. NM_202002, NM_021953, and NM_202003) andRAM2 (R1 or CDCA7L; Genbank Accession Nos. NM_018719, NM_001127370, andNM_001127371); inhibitors of apoptosis such as XIAP (Genbank AccessionNo. NM_001167); COPS signalosome subunits such as CSN1, CSN2, CSN3,CSN4, CSN5 (JAB1; Genbank Accession No. NM_006837); CSN6, CSN7A, CSN7B,and CSN8; and histone deacetylases such as HDAC1, HDAC2 (GenbankAccession No. NM_001527), HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8,HDAC9, etc.

Non-limiting examples of siRNA molecules targeting the PLK-1 geneinclude those described herein and in U.S. Patent Publication Nos.20050107316 and 20070265438; and PCT Publication No. WO 09/082817, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes. Non-limiting examples of siRNA moleculestargeting the Eg5 and XIAP genes include those described in U.S. PatentPublication No. 20090149403, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Non-limitingexamples of siRNA molecules targeting the CSN5 gene include thosedescribed in PCT Publication No. WO 09/129319, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.Non-limiting examples of siRNA molecules targeting the COP1, CSN5, RBX1,HDAC2, CDK4, WEE1, FOXM1, and RAM2 genes include those described in U.S.Provisional Application No. 61/245,143, filed Sep. 23, 2009, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

Additional examples of gene sequences associated with tumorigenesis orcell transformation include translocation sequences such as MLL fusiongenes, BCR-ABL (Wilda et al., Oncogene, 21:5716 (2002); Scherr et al.,Blood, 101:1566 (2003)), TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2,AML1-ETO, and AML1-MTG8 (Heidenreich et al., Blood, 101:3157 (2003));overexpressed sequences such as multidrug resistance genes (Nieth etal., FEBS Lett., 545:144 (2003); Wu et al, Cancer Res. 63:1515 (2003)),cyclins (Li et al., Cancer Res., 63:3593 (2003); Zou et al., Genes Dev.,16:2923 (2002)), beta-catenin (Verma et al., Clin Cancer Res., 9:1291(2003)), telomerase genes (Kosciolek et al., Mol Cancer Ther., 2:209(2003)), c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR/ErbB1(Genbank Accession Nos. NM_005228, NM_201282, NM_201283, and NM_201284;see also, Nagy et al. Exp. Cell Res., 285:39-49 (2003)), ErbB2/HER-2(Genbank Accession Nos. NM_004448 and NM_001005862), ErbB3 (GenbankAccession Nos. NM_001982 and NM_001005915), and ErbB4 (Genbank AccessionNos. NM_005235 and NM_001042599)), and mutated sequences such as RAS(Tuschl and Borkhardt, Mol. Interventions, 2:158 (2002)). Non-limitingexamples of siRNA molecules targeting the EGFR gene include thosedescribed in U.S. Patent Publication No. 20090149403, the disclosure ofwhich is herein incorporated by reference in its entirety for allpurposes. siRNA molecules that target VEGFR genes are set forth in,e.g., GB 2396864; U.S. Patent Publication No. 20040142895; and CA2456444, the disclosures of which are herein incorporated by referencein their entirety for all purposes.

Silencing of sequences that encode DNA repair enzymes find use incombination with the administration of chemotherapeutic agents (Colliset al., Cancer Res., 63:1550 (2003)). Genes encoding proteins associatedwith tumor migration are also target sequences of interest, for example,integrins, selectins, and metalloproteinases. The foregoing examples arenot exclusive. Those of skill in the art will understand that any wholeor partial gene sequence that facilitates or promotes tumorigenesis orcell transformation, tumor growth, or tumor migration can be included asa template sequence.

Angiogenic genes are able to promote the formation of new vessels.Angiogenic genes of particular interest include, but are not limited to,vascular endothelial growth factor (VEGF) (Reich et al., Mol. Vis.,9:210 (2003)), placental growth factor (PGF), VEGFR-1 (Flt-1), VEGFR-2(KDR/Flk-1), and the like. siRNA molecules that target VEGFR genes areset forth in, e.g., GB 2396864; U.S. Patent Publication No. 20040142895;and CA 2456444, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

Immunomodulator genes are genes that modulate one or more immuneresponses. Examples of immunomodulator genes include, withoutlimitation, growth factors (e.g., TGF-α, TGF-□β, EGF, FGF, IGF, NGF,PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12(Hill et al., J. Immunol., 171:691 (2003)), IL-15, IL-18, IL-20, etc.),interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), and TNF. Fas and Fasligand genes are also immunomodulator target sequences of interest (Songet al., Nat. Med., 9:347 (2003)). Genes encoding secondary signalingmolecules in hematopoietic and lymphoid cells are also included in thepresent invention, for example, Tec family kinases such as Bruton'styrosine kinase (Btk) (Heinonen et al., FEBS Lett, 527:274 (2002)).

Cell receptor ligand genes include ligands that are able to bind to cellsurface receptors (e.g., cytokine receptors, growth factor receptors,receptors with tyrosine kinase activity, G-protein coupled receptors,insulin receptor, EPO receptor, etc.) to modulate (e.g., inhibit) thephysiological pathway that the receptor is involved in (e.g., cellproliferation, tumorigenesis, cell transformation, mitogenesis, etc.).Non-limiting examples of cell receptor ligand genes include cytokines(e.g., TNF-α, interferons such as IFN-α, IFN-β, and IFN-γ, interleukinssuch as IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-17, IL-23, IL-27, chemokines, etc.), growthfactors (e.g., EGF, HB-EGF, VEGF, PEDF, SDGF, bFGF, HGF, TGF-α, TGF-β,BMP1-BMP15, PDGF, IGF, NGF, β-NGF, BDNF, NT3, NT4, GDF-9, CGF, G-CSF,GM-CSF, GDF-8, EPO, TPO, etc.), insulin, glucagon, G-protein coupledreceptor ligands, etc.

Genes associated with viral infection and survival include thoseexpressed by a host (e.g., a host factor such as tissue factor (TF)) ora virus in order to bind, enter, and replicate in a cell. Of particularinterest are viral sequences associated with chronic viral diseases.Viral sequences of particular interest include sequences of Filovirusessuch as Ebola virus and Marburg virus (see, e.g., Geisbert et al., J.Infect. Dis., 193:1650-1657 (2006)); Arenaviruses such as Lassa virus,Junin virus, Machupo virus, Guanarito virus, and Sabia virus (Buchmeieret al., Arenaviridae: the viruses and their replication, In: FIELDSVIROLOGY, Knipe et al. (eds.), 4th ed., Lippincott-Raven, Philadelphia,(2001)); Influenza viruses such as Influenza A, B, and C viruses, (see,e.g., Steinhauer et al., Annu Rev Genet., 36:305-332 (2002); and Neumannet al., J. Gen Virol., 83:2635-2662 (2002)); Hepatitis viruses (see,e.g., Hamasaki et al., FEBS Lett., 543:51 (2003); Yokota et al., EMBORep., 4:602 (2003); Schlomai et al., Hepatology, 37:764 (2003); Wilsonet al., Proc. Natl. Acad. Sci. USA, 100:2783 (2003); Kapadia et al.,Proc. Natl. Acad. Sci. USA, 100:2014 (2003); and FIELDS VIROLOGY, Knipeet al. (eds.), 4th ed., Lippincott-Raven, Philadelphia (2001)); HumanImmunodeficiency Virus (HIV) (Banerjea et al., Mol. Ther., 8:62 (2003);Song et al., J. Virol., 77:7174 (2003); Stephenson, JAMA, 289:1494(2003); Qin et al., Proc. Natl. Acad. Sci. USA, 100:183 (2003)); Herpesviruses (Jia et al., J. Virol., 77:3301 (2003)); and Human PapillomaViruses (HPV) (Hall et al., J. Virol., 77:6066 (2003); Jiang et al.,Oncogene, 21:6041 (2002)).

Exemplary Filovirus nucleic acid sequences that can be silenced include,but are not limited to, nucleic acid sequences encoding structuralproteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein(L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein(GP), VP24). Complete genome sequences for Ebola virus are set forth in,e.g., Genbank Accession Nos. NC_002549; AY769362; NC_006432; NC_004161;AY729654; AY354458; AY142960; AB050936; AF522874; AF499101; AF272001;and AF086833. Ebola virus VP24 sequences are set forth in, e.g., GenbankAccession Nos. U77385 and AY058897. Ebola virus L-pol sequences are setforth in, e.g., Genbank Accession No. X67110. Ebola virus VP40 sequencesare set forth in, e.g., Genbank Accession No. AY058896. Ebola virus NPsequences are set forth in, e.g., Genbank Accession No. AY058895. Ebolavirus GP sequences are set forth in, e.g., Genbank Accession No.AY058898; Sanchez et al., Virus Res., 29:215-240 (1993); Will et al., J.Virol., 67:1203-1210 (1993); Volchkov et al., FEBS Lett, 305:181-184(1992); and U.S. Pat. No. 6,713,069. Additional Ebola virus sequencesare set forth in, e.g., Genbank Accession Nos. L11365 and X61274.Complete genome sequences for Marburg virus are set forth in, e.g.,Genbank Accession Nos. NC_001608; AY430365; AY430366; and AY358025.Marburg virus GP sequences are set forth in, e.g., Genbank AccessionNos. AF005734; AF005733; and AF005732. Marburg virus VP35 sequences areset forth in, e.g., Genbank Accession Nos. AF005731 and AF005730.Additional Marburg virus sequences are set forth in, e.g., GenbankAccession Nos. X64406; Z29337; AF005735; and Z12132. Non-limitingexamples of siRNA molecules targeting Ebola virus and Marburg virusnucleic acid sequences include those described in U.S. PatentPublication No. 20070135370 and U.S. Provisional Application No.61/286,741, filed Dec. 15, 2009, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

Exemplary Arenavirus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences encodingnucleoprotein (NP), glycoprotein (GP), L-polymerase (L), and Z protein(Z). Complete genome sequences for Lassa virus are set forth in, e.g.,Genbank Accession Nos. NC_004296 (LASV segment S) and NC_004297 (LASVsegment L). Non-limiting examples of siRNA molecules targeting Lassavirus nucleic acid sequences include those described in U.S. ProvisionalApplication No. 61/319,855, filed Mar. 31, 2010, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

Exemplary host nucleic acid sequences that can be silenced include, butare not limited to, nucleic acid sequences encoding host factors such astissue factor (TF) that are known to play a role in the pathogenisis ofhemorrhagic fever viruses. The mRNA sequence of TF is set forth inGenbank Accession No. NM_001993. Those of skill in the art willappreciate that TF is also known as F3, coagulation factor III,thromboplastin, and CD142. Non-limiting examples of siRNA moleculestargeting TF nucleic acid sequences include those described in U.S.Provisional Application No. 61/319,855, filed Mar. 31, 2010, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

Exemplary Influenza virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences encodingnucleoprotein (NP), matrix proteins (M1 and M2), nonstructural proteins(NS1 and NS2), RNA polymerase (PA, PB1, PB2), neuraminidase (NA), andhaemagglutinin (HA). Influenza A NP sequences are set forth in, e.g.,Genbank Accession Nos. NC_004522; AY818138; AB166863; AB188817;AB189046; AB189054; AB189062; AY646169; AY646177; AY651486; AY651493;AY651494; AY651495; AY651496; AY651497; AY651498; AY651499; AY651500;AY651501; AY651502; AY651503; AY651504; AY651505; AY651506; AY651507;AY651509; AY651528; AY770996; AY790308; AY818138; and AY818140.Influenza A PA sequences are set forth in, e.g., Genbank Accession Nos.AY818132; AY790280; AY646171; AY818132; AY818133; AY646179; AY818134;AY551934; AY651613; AY651610; AY651620; AY651617; AY651600; AY651611;AY651606; AY651618; AY651608; AY651607; AY651605; AY651609; AY651615;AY651616; AY651640; AY651614; AY651612; AY651621; AY651619; AY770995;and AY724786. Non-limiting examples of siRNA molecules targetingInfluenza virus nucleic acid sequences include those described in U.S.Patent Publication No. 20070218122, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

Exemplary hepatitis virus nucleic acid sequences that can be silencedinclude, but are not limited to, nucleic acid sequences involved intranscription and translation (e.g., En1, En2, X, P) and nucleic acidsequences encoding structural proteins (e.g., core proteins including Cand C-related proteins, capsid and envelope proteins including S, M,and/or L proteins, or fragments thereof) (see, e.g., FIELDS VIROLOGY,supra). Exemplary Hepatits C virus (HCV) nucleic acid sequences that canbe silenced include, but are not limited to, the 5′-untranslated region(5′-UTR), the 3′-untranslated region (3′-UTR), the polyproteintranslation initiation codon region, the internal ribosome entry site(IRES) sequence, and/or nucleic acid sequences encoding the coreprotein, the E1 protein, the E2 protein, the p7 protein, the NS2protein, the NS3 protease/helicase, the NS4A protein, the NS4B protein,the NS5A protein, and/or the NS5B RNA-dependent RNA polymerase. HCVgenome sequences are set forth in, e.g., Genbank Accession Nos.NC_004102 (HCV genotype 1a), AJ238799 (HCV genotype 1b), NC_009823 (HCVgenotype 2), NC_009824 (HCV genotype 3), NC_009825 (HCV genotype 4),NC_009826 (HCV genotype 5), and NC_009827 (HCV genotype 6). Hepatitis Avirus nucleic acid sequences are set forth in, e.g., Genbank AccessionNo. NC_001489; Hepatitis B virus nucleic acid sequences are set forthin, e.g., Genbank Accession No. NC_003977; Hepatitis D virus nucleicacid sequence are set forth in, e.g., Genbank Accession No. NC_001653;Hepatitis E virus nucleic acid sequences are set forth in, e.g., GenbankAccession No. NC_001434; and Hepatitis G virus nucleic acid sequencesare set forth in, e.g., Genbank Accession No. NC_001710. Silencing ofsequences that encode genes associated with viral infection and survivalcan conveniently be used in combination with the administration ofconventional agents used to treat the viral condition. Non-limitingexamples of siRNA molecules targeting hepatitis virus nucleic acidsequences include those described in U.S. Patent Publication Nos.20060281175, 20050058982, and 20070149470; U.S. Pat. No. 7,348,314; andPCT Application No. PCT/CA2010/000444, entitled “Compositions andMethods for Silencing Hepatitis C Virus Expression,” filed Mar. 19,2010, bearing Attorney Docket No. 020801-008910PC, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

Genes associated with metabolic diseases and disorders (e.g., disordersin which the liver is the target and liver diseases and disorders)include, but are not limited to, genes expressed in dyslipidemia, suchas, e.g., apolipoprotein B (APOB) (Genbank Accession No. NM_000384),apolipoprotein CIII (APOC3) (Genbank Accession Nos. NM_000040 and NG008949 REGION: 5001..8164), apolipoprotein E (APOE) (Genbank AccessionNos. NM_000041 and NG 007084 REGION: 5001..8612), proprotein convertasesubtilisin/kexin type 9 (PCSK9) (Genbank Accession No. NM_174936),diacylglycerol O-acyltransferase type 1 (DGAT1) (Genbank Accession No.NM_012079), diacylglyerol O-acyltransferase type 2 (DGAT2) (GenbankAccession No. NM_032564), liver X receptors such as LXRα and LXRβ(Genback Accession No. NM_007121), farnesoid X receptors (FXR) (GenbankAccession No. NM_005123), sterol-regulatory element binding protein(SREBP), site-1 protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme-Areductase (HMG coenzyme-A reductase); and genes expressed in diabetes,such as, e.g., glucose 6-phosphatase (see, e.g., Forman et al., Cell,81:687 (1995); Seol et al., Mol. Endocrinol., 9:72 (1995), Zavacki etal., Proc. Natl. Acad. Sci. USA, 94:7909 (1997); Sakai et al., Cell,85:1037-1046 (1996); Duncan et al., J. Biol. Chem., 272:12778-12785(1997); Willy et al., Genes Dev., 9:1033-1045 (1995); Lehmann et al., J.Biol. Chem., 272:3137-3140 (1997); Janowski et al., Nature, 383:728-731(1996); and Peet et al., Cell, 93:693-704 (1998)).

One of skill in the art will appreciate that genes associated withmetabolic diseases and disorders (e.g., diseases and disorders in whichthe liver is a target and liver diseases and disorders) include genesthat are expressed in the liver itself as well as and genes expressed inother organs and tissues. Silencing of sequences that encode genesassociated with metabolic diseases and disorders can conveniently beused in combination with the administration of conventional agents usedto treat the disease or disorder. Non-limiting examples of siRNAmolecules targeting the APOB gene include those described in U.S. PatentPublication Nos. 20060134189, 20060105976, and 20070135372, and PCTPublication No. WO 04/091515, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.Non-limiting examples of siRNA molecules targeting the APOC3 geneinclude those described in PCT Application No. PCT/CA2010/000120, filedJan. 26, 2010, the disclosure of which is herein incorporated byreference in its entirety for all purposes. Non-limiting examples ofsiRNA molecules targeting the PCSK9 gene include those described in U.S.Patent Publication Nos. 20070173473, 20080113930, and 20080306015, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes. Exemplary siRNA molecules targeting the DGAT1gene may be designed using the antisense compounds described in U.S.Patent Publication No. 20040185559, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. ExemplarysiRNA molecules targeting the DGAT2 gene may be designed using theantisense compounds described in U.S. Patent Publication No.20050043524, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

Templates coding for an expansion of trinucleotide repeats (e.g., CAGrepeats) find use in silencing pathogenic sequences in neurodegenerativedisorders caused by the expansion of trinucleotide repeats, such asspinobulbular muscular atrophy and Huntington's Disease (Caplen et al.,Hum. Mol. Genet., 11:175 (2002)).

In addition to its utility in silencing the expression of any of theabove-described genes for therapeutic purposes, the siRNA describedherein are also useful in research and development applications as wellas diagnostic, prophylactic, prognostic, clinical, and other healthcareapplications. As a non-limiting example, the siRNA can be used in targetvalidation studies directed at testing whether a gene of interest hasthe potential to be a therapeutic target. The siRNA can also be used intarget identification studies aimed at discovering genes as potentialtherapeutic targets.

e) Exemplary siRNA Embodiments

In some embodiments, each strand of the siRNA molecule comprises fromabout 15 to about 60 nucleotides in length (e.g., about 15-60, 15-50,15-40, 15-30, 15-25, or 19-25 nucleotides in length, or 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25 nucleotides in length). In one particularembodiment, the siRNA is chemically synthesized. The siRNA molecules ofthe invention are capable of silencing the expression of a targetsequence in vitro and/or in vivo.

In other embodiments, the siRNA comprises at least one modifiednucleotide. In certain embodiments, the siRNA comprises one, two, three,four, five, six, seven, eight, nine, ten, or more modified nucleotidesin the double-stranded region. In particular embodiments, less thanabout 50% (e.g., less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%,10%, or 5%) of the nucleotides in the double-stranded region of thesiRNA comprise modified nucleotides. In preferred embodiments, fromabout 1% to about 50% (e.g., from about 5%-50%, 10%-50%, 15%-50%,20%-50%, 25%-50%, 30%-50%, 35%-50%, 40%-50%, 45%-50%, 5%-45%, 10%-45%,15%-45%, 20%-45%, 25%-45%, 30%-45%, 35%-45%, 40%-45%, 5%-40%, 10%-40%,15%-40%, 20%-40%, 25%-40%, 30%-40%, 35%-40%, 5%-35%, 10%-35%, 15%-35%,20%-35%, 25%-35%, 30%-35%, 5%-30%, 10%-30%, 15%-30%, 20%-30%, 25%-30%,5%-25%, 10%-25%, 15%-25%, 20%-25%, 5%-20%, 10%-20%, 15%-20%, 5%-15%,10%-15%, or 5%-10%) of the nucleotides in the double-stranded region ofthe siRNA comprise modified nucleotides.

In further embodiments, the siRNA comprises modified nucleotidesincluding, but not limited to, 2′-O-methyl (2′OMe) nucleotides,2′-deoxy-2′-fluoro (2′F) nucleotides, 2′-deoxy nucleotides,2′-O-(2-methoxyethyl) (MOE) nucleotides, locked nucleic acid (LNA)nucleotides, and mixtures thereof. In preferred embodiments, the siRNAcomprises 2′OMe nucleotides (e.g., 2′OMe purine and/or pyrimidinenucleotides) such as, e.g., 2′OMe-guanosine nucleotides, 2′OMe-uridinenucleotides, 2′OMe-adenosine nucleotides, 2′OMe-cytosine nucleotides, ormixtures thereof. In one particular embodiment, the siRNA comprises atleast one 2′OMe-guanosine nucleotide, 2′OMe-uridine nucleotide, ormixtures thereof. In certain instances, the siRNA does not comprise2′OMe-cytosine nucleotides. In other embodiments, the siRNA comprises ahairpin loop structure.

In certain embodiments, the siRNA comprises modified nucleotides in onestrand (i.e., sense or antisense) or both strands of the double-strandedregion of the siRNA molecule. Preferably, uridine and/or guanosinenucleotides are modified at selective positions in the double-strandedregion of the siRNA duplex. With regard to uridine nucleotidemodifications, at least one, two, three, four, five, six, or more of theuridine nucleotides in the sense and/or antisense strand can be amodified uridine nucleotide such as a 2′OMe-uridine nucleotide. In someembodiments, every uridine nucleotide in the sense and/or antisensestrand is a 2′OMe-uridine nucleotide. With regard to guanosinenucleotide modifications, at least one, two, three, four, five, six, ormore of the guanosine nucleotides in the sense and/or antisense strandcan be a modified guanosine nucleotide such as a 2′OMe-guanosinenucleotide. In some embodiments, every guanosine nucleotide in the senseand/or antisense strand is a 2′OMe-guanosine nucleotide.

In certain embodiments, at least one, two, three, four, five, six,seven, or more 5′-GU-3′ motifs in an siRNA sequence may be modified,e.g., by introducing mismatches to eliminate the 5′-GU-3′ motifs and/orby introducing modified nucleotides such as 2′OMe nucleotides. The5′-GU-3′ motif can be in the sense strand, the antisense strand, or bothstrands of the siRNA sequence. The 5′-GU-3′ motifs may be adjacent toeach other or, alternatively, they may be separated by 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, or more nucleotides.

In some embodiments, a modified siRNA molecule is less immunostimulatorythan a corresponding unmodified siRNA sequence. In such embodiments, themodified siRNA molecule with reduced immunostimulatory propertiesadvantageously retains RNAi activity against the target sequence. Inanother embodiment, the immunostimulatory properties of the modifiedsiRNA molecule and its ability to silence target gene expression can bebalanced or optimized by the introduction of minimal and selective 2′OMemodifications within the siRNA sequence such as, e.g., within thedouble-stranded region of the siRNA duplex. In certain instances, themodified siRNA is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% less immunostimulatory than thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that the immunostimulatory properties of the modifiedsiRNA molecule and the corresponding unmodified siRNA molecule can bedetermined by, for example, measuring INF-α and/or IL-6 levels fromabout two to about twelve hours after systemic administration in amammal or transfection of a mammalian responder cell using anappropriate lipid-based delivery system (such as the SNALP deliverysystem disclosed herein).

In other embodiments, a modified siRNA molecule has an IC₅₀ (i.e.,half-maximal inhibitory concentration) less than or equal to ten-foldthat of the corresponding unmodified siRNA (i.e., the modified siRNA hasan IC₅₀ that is less than or equal to ten-times the IC₅₀ of thecorresponding unmodified siRNA). In other embodiments, the modifiedsiRNA has an IC₅₀ less than or equal to three-fold that of thecorresponding unmodified siRNA sequence. In yet other embodiments, themodified siRNA has an IC₅₀ less than or equal to two-fold that of thecorresponding unmodified siRNA. It will be readily apparent to those ofskill in the art that a dose-response curve can be generated and theIC₅₀ values for the modified siRNA and the corresponding unmodifiedsiRNA can be readily determined using methods known to those of skill inthe art.

In another embodiment, an unmodified or modified siRNA molecule iscapable of silencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% of the expression of the target sequencerelative to a negative control (e.g., buffer only, an siRNA sequencethat targets a different gene, a scrambled siRNA sequence, etc.).

In yet another embodiment, a modified siRNA molecule is capable ofsilencing at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% of the expression of the target sequence relative tothe corresponding unmodified siRNA sequence.

In some embodiments, the siRNA molecule does not comprise phosphatebackbone modifications, e.g., in the sense and/or antisense strand ofthe double-stranded region. In other embodiments, the siRNA comprisesone, two, three, four, or more phosphate backbone modifications, e.g.,in the sense and/or antisense strand of the double-stranded region. Inpreferred embodiments, the siRNA does not comprise phosphate backbonemodifications.

In further embodiments, the siRNA does not comprise 2′-deoxynucleotides, e.g., in the sense and/or antisense strand of thedouble-stranded region. In yet further embodiments, the siRNA comprisesone, two, three, four, or more 2′-deoxy nucleotides, e.g., in the senseand/or antisense strand of the double-stranded region. In preferredembodiments, the siRNA does not comprise 2′-deoxy nucleotides.

In certain instances, the nucleotide at the 3′-end of thedouble-stranded region in the sense and/or antisense strand is not amodified nucleotide. In certain other instances, the nucleotides nearthe 3′-end (e.g., within one, two, three, or four nucleotides of the3′-end) of the double-stranded region in the sense and/or antisensestrand are not modified nucleotides.

The siRNA molecules described herein may have 3′ overhangs of one, two,three, four, or more nucleotides on one or both sides of thedouble-stranded region, or may lack overhangs (i.e., have blunt ends) onone or both sides of the double-stranded region. In certain embodiments,the 3′ overhang on the sense and/or antisense strand independentlycomprises one, two, three, four, or more modified nucleotides such as2′OMe nucleotides and/or any other modified nucleotide described hereinor known in the art.

In particular embodiments, siRNAs are administered using a carriersystem such as a nucleic acid-lipid particle. In a preferred embodiment,the nucleic acid-lipid particle comprises: (a) one or more siRNAmolecules targeting one or more genes associated with cellproliferation, tumorigenesis, and/or cell transformation (e.g., PLK-1);(b) one or more cationic lipids (e.g., one or more cationic lipids ofFormula I-XVI or salts thereof as set forth herein); (c) one or morenon-cationic lipids (e.g., DPPC, DSPC, DSPE, and/or cholesterol); and(d) one or more conjugated lipids that inhibit aggregation of theparticles (e.g., one or more PEG-lipid conjugates having an averagemolecular weight of from about 550 daltons to about 1000 daltons such asPEG750-C-DMA).

2. Dicer-Substrate dsRNA

As used herein, the term “Dicer-substrate dsRNA” or “precursor RNAimolecule” is intended to include any precursor molecule that isprocessed in vivo by Dicer to produce an active siRNA which isincorporated into the RISC complex for RNA interference of a targetgene, such as PLK-1.

In one embodiment, the Dicer-substrate dsRNA has a length sufficientsuch that it is processed by Dicer to produce an siRNA. According tothis embodiment, the Dicer-substrate dsRNA comprises (i) a firstoligonucleotide sequence (also termed the sense strand) that is betweenabout 25 and about 60 nucleotides in length (e.g., about 25-60, 25-55,25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), preferablybetween about 25 and about 30 nucleotides in length (e.g., 25, 26, 27,28, 29, or 30 nucleotides in length), and (ii) a second oligonucleotidesequence (also termed the antisense strand) that anneals to the firstsequence under biological conditions, such as the conditions found inthe cytoplasm of a cell. The second oligonucleotide sequence may bebetween about 25 and about 60 nucleotides in length (e.g., about 25-60,25-55, 25-50, 25-45, 25-40, 25-35, or 25-30 nucleotides in length), andis preferably between about 25 and about 30 nucleotides in length (e.g.,25, 26, 27, 28, 29, or 30 nucleotides in length). In addition, a regionof one of the sequences, particularly of the antisense strand, of theDicer-substrate dsRNA has a sequence length of at least about 19nucleotides, for example, from about 19 to about 60 nucleotides (e.g.,about 19-60, 19-55, 19-50, 19-45, 19-40, 19-35, 19-30, or 19-25nucleotides), preferably from about 19 to about 23 nucleotides (e.g.,19, 20, 21, 22, or 23 nucleotides) that are sufficiently complementaryto a nucleotide sequence of the RNA produced from the target gene totrigger an RNAi response.

In a second embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and has at least one of the following properties: (i)the dsRNA is asymmetric, e.g., has a 3′-overhang on the antisensestrand; and/or (ii) the dsRNA has a modified 3′-end on the sense strandto direct orientation of Dicer binding and processing of the dsRNA to anactive siRNA. According to this latter embodiment, the sense strandcomprises from about 22 to about 28 nucleotides and the antisense strandcomprises from about 24 to about 30 nucleotides.

In one embodiment, the Dicer-substrate dsRNA has an overhang on the3′-end of the antisense strand. In another embodiment, the sense strandis modified for Dicer binding and processing by suitable modifierslocated at the 3′-end of the sense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the Dicer-substrate dsRNA has an overhangon the 3′-end of the antisense strand and the sense strand is modifiedfor Dicer processing. In another embodiment, the 5′-end of the sensestrand has a phosphate. In another embodiment, the 5′-end of theantisense strand has a phosphate. In another embodiment, the antisensestrand or the sense strand or both strands have one or more 2′-O-methyl(2′OMe) modified nucleotides. In another embodiment, the antisensestrand contains 2′OMe modified nucleotides. In another embodiment, theantisense stand contains a 3′-overhang that is comprised of 2′OMemodified nucleotides. The antisense strand could also include additional2′OMe modified nucleotides. The sense and antisense strands anneal underbiological conditions, such as the conditions found in the cytoplasm ofa cell. In addition, a region of one of the sequences, particularly ofthe antisense strand, of the Dicer-substrate dsRNA has a sequence lengthof at least about 19 nucleotides, wherein these nucleotides are in the21-nucleotide region adjacent to the 3′-end of the antisense strand andare sufficiently complementary to a nucleotide sequence of the RNAproduced from the target gene, such as PLK-1. Further, in accordancewith this embodiment, the Dicer-substrate dsRNA may also have one ormore of the following additional properties: (a) the antisense strandhas a right shift from the typical 21-mer (i.e., the antisense strandincludes nucleotides on the right side of the molecule when compared tothe typical 21-mer); (b) the strands may not be completelycomplementary, i.e., the strands may contain simple mismatch pairings;and (c) base modifications such as locked nucleic acid(s) may beincluded in the 5′-end of the sense strand.

In a third embodiment, the sense strand comprises from about 25 to about28 nucleotides (e.g., 25, 26, 27, or 28 nucleotides), wherein the 2nucleotides on the 3′-end of the sense strand are deoxyribonucleotides.The sense strand contains a phosphate at the 5′-end. The antisensestrand comprises from about 26 to about 30 nucleotides (e.g., 26, 27,28, 29, or 30 nucleotides) and contains a 3′-overhang of 1-4nucleotides. The nucleotides comprising the 3′-overhang are modifiedwith 2′OMe modified ribonucleotides. The antisense strand containsalternating 2′OMe modified nucleotides beginning at the first monomer ofthe antisense strand adjacent to the 3′-overhang, and extending 15-19nucleotides from the first monomer adjacent to the 3′-overhang. Forexample, for a 27-nucleotide antisense strand and counting the firstbase at the 5′-end of the antisense strand as position number 1, 2′OMemodifications would be placed at bases 9, 11, 13, 15, 17, 19, 21, 23,25, 26, and 27. In one embodiment, the Dicer-substrate dsRNA has thefollowing structure:

5′-pXXXXXXXXXXXXXXXXXXXXXXXDD-3′ 3′-YXXXXXXXXXXXXXXXXXXXXXXXXXp-5′wherein “X”=RNA, “p”=a phosphate group, “X”=2′OMe RNA, “Y” is anoverhang domain comprised of 1, 2, 3, or 4 RNA monomers that areoptionally 2′OMe RNA monomers, and “D”=DNA. The top strand is the sensestrand, and the bottom strand is the antisense strand.

In a fourth embodiment, the Dicer-substrate dsRNA has several propertieswhich enhance its processing by Dicer. According to this embodiment, thedsRNA has a length sufficient such that it is processed by Dicer toproduce an siRNA and at least one of the following properties: (i) thedsRNA is asymmetric, e.g., has a 3′-overhang on the sense strand; and(ii) the dsRNA has a modified 3′-end on the antisense strand to directorientation of Dicer binding and processing of the dsRNA to an activesiRNA. According to this embodiment, the sense strand comprises fromabout 24 to about 30 nucleotides (e.g., 24, 25, 26, 27, 28, 29, or 30nucleotides) and the antisense strand comprises from about 22 to about28 nucleotides (e.g., 22, 23, 24, 25, 26, 27, or 28 nucleotides). In oneembodiment, the Dicer-substrate dsRNA has an overhang on the 3′-end ofthe sense strand. In another embodiment, the antisense strand ismodified for Dicer binding and processing by suitable modifiers locatedat the 3′-end of the antisense strand. Suitable modifiers includenucleotides such as deoxyribonucleotides, acyclonucleotides, and thelike, and sterically hindered molecules such as fluorescent moleculesand the like. When nucleotide modifiers are used, they replaceribonucleotides in the dsRNA such that the length of the dsRNA does notchange. In another embodiment, the dsRNA has an overhang on the 3′-endof the sense strand and the antisense strand is modified for Dicerprocessing. In one embodiment, the antisense strand has a 5′-phosphate.The sense and antisense strands anneal under biological conditions, suchas the conditions found in the cytoplasm of a cell. In addition, aregion of one of the sequences, particularly of the antisense strand, ofthe dsRNA has a sequence length of at least 19 nucleotides, whereinthese nucleotides are adjacent to the 3′-end of antisense strand and aresufficiently complementary to a nucleotide sequence of the RNA producedfrom the target gene, such as PLK-1. Further, in accordance with thisembodiment, the Dicer-substrate dsRNA may also have one or more of thefollowing additional properties: (a) the antisense strand has a leftshift from the typical 21-mer (i.e., the antisense strand includesnucleotides on the left side of the molecule when compared to thetypical 21-mer); and (b) the strands may not be completelycomplementary, i.e., the strands may contain simple mismatch pairings.

In a preferred embodiment, the Dicer-substrate dsRNA has an asymmetricstructure, with the sense strand having a 25-base pair length, and theantisense strand having a 27-base pair length with a 2 base 3′-overhang.In certain instances, this dsRNA having an asymmetric structure furthercontains 2 deoxynucleotides at the 3′-end of the sense strand in placeof two of the ribonucleotides. In certain other instances, this dsRNAhaving an asymmetric structure further contains 2′OMe modifications atpositions 9, 11, 13, 15, 17, 19, 21, 23, and 25 of the antisense strand(wherein the first base at the 5′-end of the antisense strand isposition 1). In certain additional instances, this dsRNA having anasymmetric structure further contains a 3′-overhang on the antisensestrand comprising 1, 2, 3, or 4 2′OMe nucleotides (e.g., a 3′-overhangof 2′OMe nucleotides at positions 26 and 27 on the antisense strand).

In another embodiment, Dicer-substrate dsRNAs may be designed by firstselecting an antisense strand siRNA sequence having a length of at least19 nucleotides. In some instances, the antisense siRNA is modified toinclude about 5 to about 11 ribonucleotides on the 5′-end to provide alength of about 24 to about 30 nucleotides. When the antisense strandhas a length of 21 nucleotides, 3-9, preferably 4-7, or more preferably6 nucleotides may be added on the 5′-end. Although the addedribonucleotides may be complementary to the target gene sequence, fullcomplementarity between the target sequence and the antisense siRNA isnot required. That is, the resultant antisense siRNA is sufficientlycomplementary with the target sequence. A sense strand is then producedthat has about 22 to about 28 nucleotides. The sense strand issubstantially complementary with the antisense strand to anneal to theantisense strand under biological conditions. In one embodiment, thesense strand is synthesized to contain a modified 3′-end to direct Dicerprocessing of the antisense strand. In another embodiment, the antisensestrand of the dsRNA has a 3′-overhang. In a further embodiment, thesense strand is synthesized to contain a modified 3′-end for Dicerbinding and processing and the antisense strand of the dsRNA has a3′-overhang.

In a related embodiment, the antisense siRNA may be modified to includeabout 1 to about 9 ribonucleotides on the 5′-end to provide a length ofabout 22 to about 28 nucleotides. When the antisense strand has a lengthof 21 nucleotides, 1-7, preferably 2-5, or more preferably 4ribonucleotides may be added on the 3′-end. The added ribonucleotidesmay have any sequence. Although the added ribonucleotides may becomplementary to the target gene sequence, full complementarity betweenthe target sequence and the antisense siRNA is not required. That is,the resultant antisense siRNA is sufficiently complementary with thetarget sequence. A sense strand is then produced that has about 24 toabout 30 nucleotides. The sense strand is substantially complementarywith the antisense strand to anneal to the antisense strand underbiological conditions. In one embodiment, the antisense strand issynthesized to contain a modified 3′-end to direct Dicer processing. Inanother embodiment, the sense strand of the dsRNA has a 3′-overhang. Ina further embodiment, the antisense strand is synthesized to contain amodified 3′-end for Dicer binding and processing and the sense strand ofthe dsRNA has a 3′-overhang.

Suitable Dicer-substrate dsRNA sequences can be identified, synthesized,and modified using any means known in the art for designing,synthesizing, and modifying siRNA sequences. In particular embodiments,Dicer-substrate dsRNAs are administered using a carrier system such as anucleic acid-lipid particle. In a preferred embodiment, the nucleicacid-lipid particle comprises: (a) one or more Dicer-substrate dsRNAmolecules targeting one or more genes associated with cellproliferation, tumorigenesis, and/or cell transformation (e.g., PLK-1);(b) one or more cationic lipids (e.g., one or more cationic lipids ofFormula I-XVI or salts thereof as set forth herein); (c) one or morenon-cationic lipids (e.g., DPPC, DSPC, DSPE, and/or cholesterol); and(d) one or more conjugated lipids that inhibit aggregation of theparticles (e.g., one or more PEG-lipid conjugates having an averagemolecular weight of from about 550 daltons to about 1000 daltons such asPEG750-C-DMA).

Additional embodiments related to the Dicer-substrate dsRNAs of theinvention, as well as methods of designing and synthesizing such dsRNAs,are described in U.S. Patent Publication Nos. 20050244858, 20050277610,and 20070265220, and U.S. application Ser. No. 12/794,701, filed Jun. 4,2010, the disclosures of which are herein incorporated by reference intheir entirety for all purposes.

3. Small Hairpin RNA (shRNA)

A “small hairpin RNA” or “short hairpin RNA” or “shRNA” includes a shortRNA sequence that makes a tight hairpin turn that can be used to silencegene expression via RNA interference. The shRNAs of the invention may bechemically synthesized or transcribed from a transcriptional cassette ina DNA plasmid. The shRNA hairpin structure is cleaved by the cellularmachinery into siRNA, which is then bound to the RNA-induced silencingcomplex (RISC).

The shRNAs of the invention are typically about 15-60, 15-50, or 15-40(duplex) nucleotides in length, more typically about 15-30, 15-25, or19-25 (duplex) nucleotides in length, and are preferably about 20-24,21-22, or 21-23 (duplex) nucleotides in length (e.g., each complementarysequence of the double-stranded shRNA is 15-60, 15-50, 15-40, 15-30,15-25, or 19-25 nucleotides in length, preferably about 20-24, 21-22, or21-23 nucleotides in length, and the double-stranded shRNA is about15-60, 15-50, 15-40, 15-30, 15-25, or 19-25 base pairs in length,preferably about 18-22, 19-20, or 19-21 base pairs in length). shRNAduplexes may comprise 3′ overhangs of about 1 to about 4 nucleotides orabout 2 to about 3 nucleotides on the antisense strand and/or5′-phosphate termini on the sense strand. In some embodiments, the shRNAcomprises a sense strand and/or antisense strand sequence of from about15 to about 60 nucleotides in length (e.g., about 15-60, 15-55, 15-50,15-45, 15-40, 15-35, 15-30, or 15-25 nucleotides in length), preferablyfrom about 19 to about 40 nucleotides in length (e.g., about 19-40,19-35, 19-30, or 19-25 nucleotides in length), more preferably fromabout 19 to about 23 nucleotides in length (e.g., 19, 20, 21, 22, or 23nucleotides in length).

Non-limiting examples of shRNA include a double-stranded polynucleotidemolecule assembled from a single-stranded molecule, where the sense andantisense regions are linked by a nucleic acid-based or non-nucleicacid-based linker; and a double-stranded polynucleotide molecule with ahairpin secondary structure having self-complementary sense andantisense regions. In preferred embodiments, the sense and antisensestrands of the shRNA are linked by a loop structure comprising fromabout 1 to about 25 nucleotides, from about 2 to about 20 nucleotides,from about 4 to about 15 nucleotides, from about 5 to about 12nucleotides, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, or more nucleotides.

Additional shRNA sequences include, but are not limited to, asymmetricshRNA precursor polynucleotides such as those described in PCTPublication Nos. WO 2006/074108 and WO 2009/076321, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. For example, PCT Publication No. WO 2006/074108 disclosesself-protected oligonucleotides comprising a region having a sequencecomplementary to one, two, three, or more same or different target mRNAsequences (e.g., multivalent shRNAs) and one or more self-complementaryregions. Similarly, PCT Publication No. WO 2009/076321 disclosesself-forming asymmetric precursor polynucleotides comprising a targetingregion comprising a polynucleotide sequence complementary to a region ofone, two, three, or more same or different target mRNA sequences (e.g.,multivalent shRNAs); a first self-complementary region; and a secondself-complementary region, wherein the first and secondself-complementary regions are located one at each end of the targetingregion and both self-complementary regions form stem-loop structures,wherein the first self-complementary region is capable of being cleavedby a RNase III endoribonuclease that is not a class IV DICERendoribonuclease, and wherein both self-complementary regions comprise anucleotide sequence that is complementary to a region of the target genesequence, but wherein a portion of the target sequence present in thetargeting region does not have a complementary sequence in either of theself-complementary regions.

Suitable shRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. In particular embodiments, shRNAs areadministered using a carrier system such as a nucleic acid-lipidparticle. In a preferred embodiment, the nucleic acid-lipid particlecomprises: (a) one or more shRNA molecules targeting one or more genesassociated with cell proliferation, tumorigenesis, and/or celltransformation (e.g., PLK-1); (b) one or more cationic lipids (e.g., oneor more cationic lipids of Formula I-XVI or salts thereof as set forthherein); (c) one or more non-cationic lipids (e.g., DPPC, DSPC, DSPE,and/or cholesterol); and (d) one or more conjugated lipids that inhibitaggregation of the particles (e.g., one or more PEG-lipid conjugateshaving an average molecular weight of from about 550 daltons to about1000 daltons such as PEG750-C-DMA).

Additional embodiments related to the shRNAs of the invention, as wellas methods of designing and synthesizing such shRNAs, are described inU.S. patent application Ser. No. 12/794,701, filed Jun. 4, 2010, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes.

4. aiRNA

Like siRNA, asymmetrical interfering RNA (aiRNA) can recruit theRNA-induced silencing complex (RISC) and lead to effective silencing ofa variety of genes in mammalian cells by mediating sequence-specificcleavage of the target sequence between nucleotide 10 and 11 relative tothe 5′ end of the antisense strand (Sun et al., Nat. Biotech.,26:1379-1382 (2008)). Typically, an aiRNA molecule comprises a short RNAduplex having a sense strand and an antisense strand, wherein the duplexcontains overhangs at the 3′ and 5′ ends of the antisense strand. TheaiRNA is generally asymmetric because the sense strand is shorter onboth ends when compared to the complementary antisense strand. In someaspects, aiRNA molecules may be designed, synthesized, and annealedunder conditions similar to those used for siRNA molecules. As anon-limiting example, aiRNA sequences may be selected and generatedusing the methods described above for selecting siRNA sequences.

In another embodiment, aiRNA duplexes of various lengths (e.g., about10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs) may be designed withoverhangs at the 3′ and 5′ ends of the antisense strand to target anmRNA of interest. In certain instances, the sense strand of the aiRNAmolecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or20 nucleotides in length. In certain other instances, the antisensestrand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and is preferably about 20-24, 21-22, or 21-23 nucleotides inlength.

In some embodiments, the 5′ antisense overhang contains one, two, three,four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”, etc.).In other embodiments, the 3′ antisense overhang contains one, two,three, four, or more nontargeting nucleotides (e.g., “AA”, “UU”, “dTdT”,etc.). In certain aspects, the aiRNA molecules described herein maycomprise one or more modified nucleotides, e.g., in the double-stranded(duplex) region and/or in the antisense overhangs. As a non-limitingexample, aiRNA sequences may comprise one or more of the modifiednucleotides described above for siRNA sequences. In a preferredembodiment, the aiRNA molecule comprises 2′OMe nucleotides such as, forexample, 2′OMe-guanosine nucleotides, 2′OMe-uridine nucleotides, ormixtures thereof.

In certain embodiments, aiRNA molecules may comprise an antisense strandwhich corresponds to the antisense strand of an siRNA molecule, e.g.,one of the siRNA molecules described herein. In particular embodiments,aiRNAs are administered using a carrier system such as a nucleicacid-lipid particle. In a preferred embodiment, the nucleic acid-lipidparticle comprises: (a) one or more aiRNA molecules targeting one ormore genes associated with cell proliferation, tumorigenesis, and/orcell transformation (e.g., PLK-1); (b) one or more cationic lipids(e.g., one or more cationic lipids of Formula I-XVI or salts thereof asset forth herein); (c) one or more non-cationic lipids (e.g., DPPC,DSPC, DSPE, and/or cholesterol); and (d) one or more conjugated lipidsthat inhibit aggregation of the particles (e.g., one or more PEG-lipidconjugates having an average molecular weight of from about 550 daltonsto about 1000 daltons such as PEG750-C-DMA).

Suitable aiRNA sequences can be identified, synthesized, and modifiedusing any means known in the art for designing, synthesizing, andmodifying siRNA sequences. Additional embodiments related to the aiRNAmolecules of the invention are described in U.S. Patent Publication No.20090291131 and PCT Publication No. WO 09/127060, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

5. miRNA

Generally, microRNAs (miRNA) are single-stranded RNA molecules of about21-23 nucleotides in length which regulate gene expression. miRNAs areencoded by genes from whose DNA they are transcribed, but miRNAs are nottranslated into protein (non-coding RNA); instead, each primarytranscript (a pri-miRNA) is processed into a short stem-loop structurecalled a pre-miRNA and finally into a functional mature miRNA. MaturemiRNA molecules are either partially or completely complementary to oneor more messenger RNA (mRNA) molecules, and their main function is todownregulate gene expression. The identification of miRNA molecules isdescribed, e.g., in Lagos-Quintana et al., Science, 294:853-858; Lau etal., Science, 294:858-862; and Lee et al., Science, 294: 862-864.

The genes encoding miRNA are much longer than the processed mature miRNAmolecule. miRNA are first transcribed as primary transcripts orpri-miRNA with a cap and poly-A tail and processed to short,˜70-nucleotide stem-loop structures known as pre-miRNA in the cellnucleus. This processing is performed in animals by a protein complexknown as the Microprocessor complex, consisting of the nuclease Droshaand the double-stranded RNA binding protein Pasha (Denli et al., Nature,432:231-235 (2004)). These pre-miRNA are then processed to mature miRNAin the cytoplasm by interaction with the endonuclease Dicer, which alsoinitiates the formation of the RNA-induced silencing complex (RISC)(Bernstein et al., Nature, 409:363-366 (2001). Either the sense strandor antisense strand of DNA can function as templates to give rise tomiRNA.

When Dicer cleaves the pre-miRNA stem-loop, two complementary short RNAmolecules are formed, but only one is integrated into the RISC complex.This strand is known as the guide strand and is selected by theargonaute protein, the catalytically active RNase in the RISC complex,on the basis of the stability of the 5′ end (Preall et al., Curr. Biol.,16:530-535 (2006)). The remaining strand, known as the anti-guide orpassenger strand, is degraded as a RISC complex substrate (Gregory etal., Cell, 123:631-640 (2005)). After integration into the active RISCcomplex, miRNAs base pair with their complementary mRNA molecules andinduce target mRNA degradation and/or translational silencing.

Mammalian miRNA molecules are usually complementary to a site in the 3′UTR of the target mRNA sequence. In certain instances, the annealing ofthe miRNA to the target mRNA inhibits protein translation by blockingthe protein translation machinery. In certain other instances, theannealing of the miRNA to the target mRNA facilitates the cleavage anddegradation of the target mRNA through a process similar to RNAinterference (RNAi). miRNA may also target methylation of genomic siteswhich correspond to targeted mRNA. Generally, miRNA function inassociation with a complement of proteins collectively termed the miRNP.

In certain aspects, the miRNA molecules described herein are about15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotidesin length, more typically about 15-30, 15-25, or 19-25 nucleotides inlength, and are preferably about 20-24, 21-22, or 21-23 nucleotides inlength. In certain other aspects, miRNA molecules may comprise one ormore modified nucleotides. As a non-limiting example, miRNA sequencesmay comprise one or more of the modified nucleotides described above forsiRNA sequences. In a preferred embodiment, the miRNA molecule comprises2′OMe nucleotides such as, for example, 2′OMe-guanosine nucleotides,2′OMe-uridine nucleotides, or mixtures thereof.

In some embodiments, miRNA molecules may be used to silence theexpression of any of the target genes described above for siRNAsequences, and preferably silence genes associated with cellproliferation, tumorigenesis, and/or cell transformation. In particularembodiments, miRNAs are administered using a carrier system such as anucleic acid-lipid particle. In a preferred embodiment, the nucleicacid-lipid particle comprises: (a) one or more miRNA molecules targetingone or more genes associated with cell proliferation, tumorigenesis,and/or cell transformation (e.g., PLK-1); (b) one or more cationiclipids (e.g., one or more cationic lipids of Formula I-XVI or saltsthereof as set forth herein); (c) one or more non-cationic lipids (e.g.,DPPC, DSPC, DSPE, and/or cholesterol); and (d) one or more conjugatedlipids that inhibit aggregation of the particles (e.g., one or morePEG-lipid conjugates having an average molecular weight of from about550 daltons to about 1000 daltons such as PEG750-C-DMA).

In other embodiments, one or more agents that block the activity of anmiRNA targeting an mRNA of interest (e.g., PLK-1 mRNA) are administeredusing a lipid particle of the invention (e.g., a nucleic acid-lipidparticle such as SNALP). Examples of blocking agents include, but arenot limited to, steric blocking oligonucleotides, locked nucleic acidoligonucleotides, and Morpholino oligonucleotides. Such blocking agentsmay bind directly to the miRNA or to the miRNA binding site on thetarget mRNA.

Additional embodiments related to the miRNA molecules of the inventionare described in U.S. Patent Publication No. 20090291131 and PCTPublication No. WO 09/127060, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

6. Antisense Oligonucleotides

In one embodiment, the nucleic acid is an antisense oligonucleotidedirected to a target gene or sequence of interest. The terms “antisenseoligonucleotide” or “antisense” include oligonucleotides that arecomplementary to a targeted polynucleotide sequence. Antisenseoligonucleotides are single strands of DNA or RNA that are complementaryto a chosen sequence. Antisense RNA oligonucleotides prevent thetranslation of complementary RNA strands by binding to the RNA.Antisense DNA oligonucleotides can be used to target a specific,complementary (coding or non-coding) RNA. If binding occurs, thisDNA/RNA hybrid can be degraded by the enzyme RNase H. In a particularembodiment, antisense oligonucleotides comprise from about 10 to about60 nucleotides, more preferably from about 15 to about 30 nucleotides.The term also encompasses antisense oligonucleotides that may not beexactly complementary to the desired target gene. Thus, the inventioncan be utilized in instances where non-target specific-activities arefound with antisense, or where an antisense sequence containing one ormore mismatches with the target sequence is the most preferred for aparticular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(see, U.S. Pat. Nos. 5,739,119 and 5,759,829). Furthermore, examples ofantisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDR1), ICAM-1, E-selectin,STK-1, striatal GABAA receptor, and human EGF (see, Jaskulski et al.,Science, 240:1544-6 (1988); Vasanthakumar et al., Cancer Commun.,1:225-32 (1989); Penis et al., Brain Res Mol Brain Res., 15; 57:310-20(1998); and U.S. Pat. Nos. 5,801,154; 5,789,573; 5,718,709 and5,610,288). Moreover, antisense constructs have also been described thatinhibit and can be used to treat a variety of abnormal cellularproliferations, e.g., cancer (see, U.S. Pat. Nos. 5,747,470; 5,591,317;and 5,783,683). The disclosures of these references are hereinincorporated by reference in their entirety for all purposes.

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v. 4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

7. Ribozymes

According to another embodiment of the invention, nucleic acid-lipidparticles are associated with ribozymes. Ribozymes are RNA-proteincomplexes having specific catalytic domains that possess endonucleaseactivity (see, Kim et al., Proc. Natl. Acad. Sci. USA., 84:8788-92(1987); and Forster et al., Cell, 49:211-20 (1987)). For example, alarge number of ribozymes accelerate phosphoester transfer reactionswith a high degree of specificity, often cleaving only one of severalphosphoesters in an oligonucleotide substrate (see, Cech et al., Cell,27:487-96 (1981); Michel et al., J. Mol. Biol., 216:585-610 (1990);Reinhold-Hurek et al., Nature, 357:173-6 (1992)). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAmolecules are known presently. Each can catalyze the hydrolysis of RNAphosphodiester bonds in trans (and thus can cleave other RNA molecules)under physiological conditions. In general, enzymatic nucleic acids actby first binding to a target RNA. Such binding occurs through the targetbinding portion of an enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, hepatitis δ virus, group I intron or RNaseP RNA (in associationwith an RNA guide sequence), or Neurospora VS RNA motif, for example.Specific examples of hammerhead motifs are described in, e.g., Rossi etal., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifsare described in, e.g., EP 0360257, Hampel et al., Biochemistry,28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990);and U.S. Pat. No. 5,631,359. An example of the hepatitis δ virus motifis described in, e.g., Perrotta et al., Biochemistry, 31:11843-52(1992). An example of the RNaseP motif is described in, e.g.,Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of theNeurospora VS RNA ribozyme motif is described in, e.g., Saville et al.,Cell, 61:685-96 (1990); Saville et al., Proc. Natl. Acad. Sci. USA,88:8826-30 (1991); Collins et al., Biochemistry, 32:2795-9 (1993). Anexample of the Group I intron is described in, e.g., U.S. Pat. No.4,987,071. Important characteristics of enzymatic nucleic acid moleculesused according to the invention are that they have a specific substratebinding site which is complementary to one or more of the target geneDNA or RNA regions, and that they have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule. Thus, the ribozyme constructs need not belimited to specific motifs mentioned herein. The disclosures of thesereferences are herein incorporated by reference in their entirety forall purposes.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in, e.g.,PCT Publication Nos. WO 93/23569 and WO 94/02595, and synthesized to betested in vitro and/or in vivo as described therein. The disclosures ofthese PCT publications are herein incorporated by reference in theirentirety for all purposes.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases(see, e.g., PCT Publication Nos. WO 92/07065, WO 93/15187, WO 91/03162,and WO 94/13688; EP 92110298.4; and U.S. Pat. No. 5,334,711, whichdescribe various chemical modifications that can be made to the sugarmoieties of enzymatic RNA molecules, the disclosures of which are eachherein incorporated by reference in their entirety for all purposes),modifications which enhance their efficacy in cells, and removal of stemII bases to shorten RNA synthesis times and reduce chemicalrequirements.

8. Immunostimulatory Oligonucleotides

Nucleic acids associated with lipid particles of the present inventionmay be immunostimulatory, including immunostimulatory oligonucleotides(ISS; single- or double-stranded) capable of inducing an immune responsewhen administered to a subject, which may be a mammal such as a human.ISS include, e.g., certain palindromes leading to hairpin secondarystructures (see, Yamamoto et al., J. Immunol., 148:4072-6 (1992)), orCpG motifs, as well as other known ISS features (such as multi-Gdomains; see; PCT Publication No. WO 96/11266, the disclosure of whichis herein incorporated by reference in its entirety for all purposes).

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target sequence in order to provoke an immuneresponse. Thus, certain immunostimulatory nucleic acids may comprise asequence corresponding to a region of a naturally-occurring gene ormRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein the CpG dinucleotide is methylated. In an alternative embodiment, thenucleic acid comprises at least two CpG dinucleotides, wherein at leastone cytosine in the CpG dinucleotides is methylated. In a furtherembodiment, each cytosine in the CpG dinucleotides present in thesequence is methylated. In another embodiment, the nucleic acidcomprises a plurality of CpG dinucleotides, wherein at least one of theCpG dinucleotides comprises a methylated cytosine. Examples ofimmunostimulatory oligonucleotides suitable for use in the compositionsand methods of the present invention are described in PCT PublicationNos. WO 02/069369, WO 01/15726, and WO 09/086558; U.S. Pat. No.6,406,705; and Raney et al., J. Pharm. Exper. Ther., 298:1185-92 (2001),the disclosures of which are herein incorporated by reference in theirentirety for all purposes. In certain embodiments, the oligonucleotidesused in the compositions and methods of the invention have aphosphodiester (“PO”) backbone or a phosphorothioate (“PS”) backbone,and/or at least one methylated cytosine residue in a CpG motif.

B. Other Active Agents

In certain embodiments, the active agent associated with the lipidparticles of the invention may comprise one or more therapeuticproteins, polypeptides, or small organic molecules or compounds.Non-limiting examples of such therapeutically effective agents or drugsinclude oncology drugs (e.g., chemotherapy drugs, hormonal therapaeuticagents, immunotherapeutic agents, radiotherapeutic agents, etc.),lipid-lowering agents, anti-viral drugs, anti-inflammatory compounds,antidepressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs such asanti-arrhythmic agents, hormones, vasoconstrictors, and steroids. Theseactive agents may be administered alone in the lipid particles of theinvention, or in combination (e.g., co-administered) with lipidparticles of the invention comprising nucleic acid such as interferingRNA.

Non-limiting examples of chemotherapy drugs include platinum-based drugs(e.g., oxaliplatin, cisplatin, carboplatin, spiroplatin, iproplatin,satraplatin, etc.), alkylating agents (e.g., cyclophosphamide,ifosfamide, chlorambucil, busulfan, melphalan, mechlorethamine,uramustine, thiotepa, nitrosoureas, etc.), anti-metabolites (e.g.,5-fluorouracil (5-FU), azathioprine, methotrexate, leucovorin,capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine,pemetrexed, raltitrexed, etc.), plant alkaloids (e.g., vincristine,vinblastine, vinorelbine, vindesine, podophyllotoxin, paclitaxel(taxol), docetaxel, etc.), topoisomerase inhibitors (e.g., irinotecan(CPT-11; Camptosar), topotecan, amsacrine, etoposide (VP16), etoposidephosphate, teniposide, etc.), antitumor antibiotics (e.g., doxorubicin,adriamycin, daunorubicin, epirubicin, actinomycin, bleomycin, mitomycin,mitoxantrone, plicamycin, etc.), tyrosine kinase inhibitors (e.g.,gefitinib (Iressa®), sunitinib (Sutent®; SU11248), erlotinib (Tarceva®;OSI-1774), lapatinib (GW572016; GW2016), canertinib (CI 1033), semaxinib(SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), imatinib(Gleevec®; STI571), dasatinib (BMS-354825), leflunomide (SU101),vandetanib (Zactima™; ZD6474), etc.), pharmaceutically acceptable saltsthereof, stereoisomers thereof, derivatives thereof, analogs thereof,and combinations thereof.

Examples of conventional hormonal therapeutic agents include, withoutlimitation, steroids (e.g., dexamethasone), finasteride, aromataseinhibitors, tamoxifen, and goserelin as well as othergonadotropin-releasing hormone agonists (GnRH).

Examples of conventional immunotherapeutic agents include, but are notlimited to, immunostimulants (e.g., Bacillus Calmette-Guérin (BCG),levamisole, interleukin-2, alpha-interferon, etc.), monoclonalantibodies (e.g., anti-CD20, anti-HER2, anti-CD52, anti-HLA-DR, andanti-VEGF monoclonal antibodies), immunotoxins (e.g., anti-CD33monoclonal antibody-calicheamicin conjugate, anti-CD22 monoclonalantibody-pseudomonas exotoxin conjugate, etc.), and radioimmunotherapy(e.g., anti-CD20 monoclonal antibody conjugated to ¹¹¹In, ⁹⁰Y, or ¹³¹I,etc.).

Examples of conventional radiotherapeutic agents include, but are notlimited to, radionuclides such as ⁴⁷Sc, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y,¹⁰⁵Rh, ¹¹¹Ag, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re,¹⁸⁸Re, ²¹¹At, and ²¹²Bi, optionally conjugated to antibodies directedagainst tumor antigens.

Additional oncology drugs that may be used according to the inventioninclude, but are not limited to, alkeran, allopurinol, altretamine,amifostine, anastrozole, araC, arsenic trioxide, bexarotene, biCNU,carmustine, CCNU, celecoxib, cladribine, cyclosporin A, cytosinearabinoside, cytoxan, dexrazoxane, DTIC, estramustine, exemestane,FK506, gemtuzumab-ozogamicin, hydrea, hydroxyurea, idarubicin,interferon, letrozole, leustatin, leuprolide, litretinoin, megastrol,L-PAM, mesna, methoxsalen, mithramycin, nitrogen mustard, pamidronate,Pegademase, pentostatin, porfimer sodium, prednisone, rituxan,streptozocin, STI-571, taxotere, temozolamide, VM-26, toremifene,tretinoin, ATRA, valrubicin, and velban. Other examples of oncologydrugs that may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors, and camptothecins.

Non-limiting examples of lipid-lowering agents for treating a lipiddisease or disorder associated with elevated triglycerides, cholesterol,and/or glucose include statins, fibrates, ezetimibe, thiazolidinediones,niacin, beta-blockers, nitroglycerin, calcium antagonists, fish oil, andmixtures thereof.

Examples of anti-viral drugs include, but are not limited to, abacavir,aciclovir, acyclovir, adefovir, amantadine, amprenavir, arbidol,atazanavir, atripla, cidofovir, combivir, darunavir, delavirdine,didanosine, docosanol, edoxudine, efavirenz, emtricitabine, enfuvirtide,entecavir, entry inhibitors, famciclovir, fixed dose combinations,fomivirsen, fosamprenavir, foscarnet, fosfonet, fusion inhibitors,ganciclovir, ibacitabine, imunovir, idoxuridine, imiquimod, indinavir,inosine, integrase inhibitors, interferon type III (e.g., IFN-λmolecules such as IFN-λ1, IFN-λ2, and IFN-λ3), interferon type II (e.g.,IFN-γ), interferon type I (e.g., IFN-α such as PEGylated IFN-α, IFN-β,IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ), interferon, lamivudine,lopinavir, loviride, MK-0518, maraviroc, moroxydine, nelfinavir,nevirapine, nexavir, nucleoside analogues, oseltamivir, penciclovir,peramivir, pleconaril, podophyllotoxin, protease inhibitors, reversetranscriptase inhibitors, ribavirin, rimantadine, ritonavir, saquinavir,stavudine, synergistic enhancers, tenofovir, tenofovir disoproxil,tipranavir, trifluridine, trizivir, tromantadine, truvada, valaciclovir,valganciclovir, vicriviroc, vidarabine, viramidine, zalcitabine,zanamivir, zidovudine, pharmaceutically acceptable salts thereof,stereoisomers thereof, derivatives thereof, analogs thereof, andmixtures thereof.

V. Lipid Particles

In certain aspects, the present invention provides lipid particlescomprising one or more of the cationic (amino) lipids or salts thereofdescribed herein. In some embodiments, the lipid particles of theinvention further comprise one or more non-cationic lipids. In otherembodiments, the lipid particles further comprise one or more conjugatedlipids capable of reducing or inhibiting particle aggregation. Inadditional embodiments, the lipid particles further comprise one or moreactive agents or therapeutic agents such as therapeutic nucleic acids(e.g., interfering RNA such as siRNA).

The lipid particles of the present invention have a non-lamellarmorphology, i.e., a non-bilayer structure. More particularly, thepresent invention provides a composition comprising a plurality ofnucleic acid-lipid particles, wherein each particle in the plurality ofparticles comprises: (a) a nucleic acid; (b) a cationic lipid comprisingfrom about 50 mol % to about 85 mol % of the total lipid present in theparticle; (c) a non-cationic lipid comprising from about 13 mol % toabout 49.5 mol % of the total lipid present in the particle; and (d) aconjugated lipid that inhibits aggregation of particles comprising fromabout 0.5 mol % to about 10 mol % of the total lipid present in theparticle, wherein at least about 95% of the particles in the pluralityof particles have a non-lamellar morphology. In preferred embodiments,greater than 95%, preferably, greater than 96%, preferably, greater than97%, preferably, greater than 98% and, preferably, greater than 99% ofthe particles have a non-lamellar morphology, i.e., a non-bilayerstructure.

The lipid particles of the invention typically comprise an active agentor therapeutic agent, a cationic lipid, a non-cationic lipid, and aconjugated lipid that inhibits aggregation of particles. In someembodiments, the active agent or therapeutic agent is fully encapsulatedwithin the lipid portion of the lipid particle such that the activeagent or therapeutic agent in the lipid particle is resistant in aqueoussolution to enzymatic degradation, e.g., by a nuclease or protease. Inother embodiments, the lipid particles described herein aresubstantially non-toxic to mammals such as humans. The lipid particlesof the invention typically have a mean diameter of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, from about 70 to about 90 nm, from about 40 nm to about 90nm, from about 45 nm to about 85, or from about 50 nm to abut 80 nm. Thelipid particles of the invention also typically have a lipid:therapeuticagent (e.g., lipid:nucleic acid) ratio (mass/mass ratio) of from about1:1 to about 100:1, from about 1:1 to about 50:1, from about 2:1 toabout 25:1, from about 3:1 to about 20:1, from about 5:1 to about 15:1,or from about 5:1 to about 10:1.

In preferred embodiments, the lipid particles of the invention areserum-stable nucleic acid-lipid particles (SNALP) which comprise aninterfering RNA (e.g., siRNA, Dicer-substrate dsRNA, shRNA, aiRNA,and/or miRNA), a cationic lipid (e.g., one or more cationic lipids ofFormula I or salts thereof as set forth herein), a non-cationic lipid(e.g., mixtures of one or more phospholipids and cholesterol), and aconjugated lipid that inhibits aggregation of the particles (e.g., oneor more PEG-lipid and/or POZ-lipid conjugates). The SNALP may compriseat least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodified and/ormodified interfering RNA molecules. Nucleic acid-lipid particles andtheir method of preparation are described in, e.g., U.S. Pat. Nos.5,753,613; 5,785,992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; and6,320,017; and PCT Publication No. WO 96/40964, the disclosures of whichare each herein incorporated by reference in their entirety for allpurposes.

In the nucleic acid-lipid particles of the invention, the nucleic acidmay be fully encapsulated within the lipid portion of the particle,thereby protecting the nucleic acid from nuclease degradation. Inpreferred embodiments, a SNALP comprising a nucleic acid such as aninterfering RNA is fully encapsulated within the lipid portion of theparticle, thereby protecting the nucleic acid from nuclease degradation.In certain instances, the nucleic acid in the SNALP is not substantiallydegraded after exposure of the particle to a nuclease at 37° C. for atleast about 20, 30, 45, or 60 minutes. In certain other instances, thenucleic acid in the SNALP is not substantially degraded after incubationof the particle in serum at 37° C. for at least about 30, 45, or 60minutes or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18,20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In other embodiments, thenucleic acid is complexed with the lipid portion of the particle. One ofthe benefits of the formulations of the present invention is that thenucleic acid-lipid particle compositions are substantially non-toxic tomammals such as humans.

The term “fully encapsulated” indicates that the nucleic acid in thenucleic acid-lipid particle is not significantly degraded after exposureto serum or a nuclease assay that would significantly degrade free DNAor RNA. In a fully encapsulated system, preferably less than about 25%of the nucleic acid in the particle is degraded in a treatment thatwould normally degrade 100% of free nucleic acid, more preferably lessthan about 10%, and most preferably less than about 5% of the nucleicacid in the particle is degraded. “Fully encapsulated” also indicatesthat the nucleic acid-lipid particles are serum-stable, that is, thatthey do not rapidly decompose into their component parts upon in vivoadministration.

In the context of nucleic acids, full encapsulation may be determined byperforming a membrane-impermeable fluorescent dye exclusion assay, whichuses a dye that has enhanced fluorescence when associated with nucleicacid. Specific dyes such as OliGreen® and RiboGreen® (Invitrogen Corp.;Carlsbad, Calif.) are available for the quantitative determination ofplasmid DNA, single-stranded deoxyribonucleotides, and/or single- ordouble-stranded ribonucleotides. Encapsulation is determined by addingthe dye to a liposomal formulation, measuring the resultingfluorescence, and comparing it to the fluorescence observed uponaddition of a small amount of nonionic detergent. Detergent-mediateddisruption of the liposomal bilayer releases the encapsulated nucleicacid, allowing it to interact with the membrane-impermeable dye. Nucleicacid encapsulation may be calculated as E=(I_(o)−I)/I_(o), where I andI_(o) refer to the fluorescence intensities before and after theaddition of detergent (see, Wheeler et al., Gene Ther., 6:271-281(1999)).

In other embodiments, the present invention provides a nucleicacid-lipid particle (e.g., SNALP) composition comprising a plurality ofnucleic acid-lipid particles.

In some instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the particles have the nucleic acid encapsulated therein.

In other instances, the SNALP composition comprises nucleic acid that isfully encapsulated within the lipid portion of the particles, such thatfrom about 30% to about 100%, from about 40% to about 100%, from about50% to about 100%, from about 60% to about 100%, from about 70% to about100%, from about 80% to about 100%, from about 90% to about 100%, fromabout 30% to about 95%, from about 40% to about 95%, from about 50% toabout 95%, from about 60% to about 95%, from about 70% to about 95%,from about 80% to about 95%, from about 85% to about 95%, from about 90%to about 95%, from about 30% to about 90%, from about 40% to about 90%,from about 50% to about 90%, from about 60% to about 90%, from about 70%to about 90%, from about 80% to about 90%, or at least about 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% (or any fraction thereof or rangetherein) of the input nucleic acid is encapsulated in the particles.

Depending on the intended use of the lipid particles of the invention,the proportions of the components can be varied and the deliveryefficiency of a particular formulation can be measured using, e.g., anendosomal release parameter (ERP) assay.

In particular embodiments, the present invention provides a lipidparticle (e.g., SNALP) composition comprising a plurality of lipidparticles described herein and an antioxidant. In certain instances, theantioxidant in the lipid particle composition reduces, prevents, and/orinhibits the degradation of a cationic lipid present in the lipidparticle. In instances wherein the active agent is a therapeutic nucleicacid such as an interfering RNA (e.g., siRNA), the antioxidant in thelipid particle composition reduces, prevents, and/or inhibits thedegradation of the nucleic acid payload, e.g., by reducing, preventing,and/or inhibiting the formation of adducts between the nucleic acid andthe cationic lipid. Non-limiting examples of antioxidants includehydrophilic antioxidants such as chelating agents (e.g., metal chelatorssuch as ethylenediaminetetraacetic acid (EDTA), citrate, and the like),lipophilic antioxidants (e.g., vitamin E isomers, polyphenols, and thelike), salts thereof; and mixtures thereof. If needed, the antioxidantis typically present in an amount sufficient to prevent, inhibit, and/orreduce the degradation of the cationic lipid and/or active agent presentin the particle, e.g., at least about 20 mM EDTA or a salt thereof, orat least about 100 mM citrate or a salt thereof. An antioxidant such asEDTA and/or citrate may be included at any step or at multiple steps inthe lipid particle formation process described in Section VI (e.g.,prior to, during, and/or after lipid particle formation).

Additional embodiments related to methods of preventing the degradationof cationic lipids and/or active agents (e.g., therapeutic nucleicacids) present in lipid particles, compositions comprising lipidparticles stabilized by these methods, methods of making these lipidparticles, and methods of delivering and/or administering these lipidparticles are described in U.S. Provisional Application No. 61/265,671,entitled “SNALP Formulations Containing Antioxidants,” filed Dec. 1,2009, the disclosure of which is herein incorporated by reference in itsentirety for all purposes.

A. Cationic Lipids

Any of the novel cationic lipids of Formula I or salts thereof as setforth herein may be used in the lipid particles of the present invention(e.g., SNALP), either alone or in combination with one or more othercationic lipid species or non-cationic lipid species.

1. Novel Cationic Lipids

The present invention provides, inter alia, novel cationic (amino)lipids that can advantageously be used in the lipid particles describedherein for the in vitro and/or in vivo delivery of therapeutic agentssuch as nucleic acids to cells. The novel cationic lipids of the presentinvention have the structure set forth in Formula I herein, and includethe (R) and/or (S) enantiomers thereof.

In some embodiments, a lipid of the present invention comprises aracemic mixture. In other embodiments, a lipid of the present inventioncomprises a mixture of one or more diastereomers. In certainembodiments, a lipid of the present invention is enriched in oneenantiomer, such that the lipid comprises at least about 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, or 95% enantiomeric excess. In certain otherembodiments, a lipid of the present invention is enriched in onediastereomer, such that the lipid comprises at least about 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, or 95% diastereomeric excess. In certainadditional embodiments, a lipid of the present invention is chirallypure (e.g., comprises a single optical isomer). In further embodiments,a lipid of the present invention is enriched in one optical isomer(e.g., an optically active isomer), such that the lipid comprises atleast about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% isomericexcess. The present invention provides the synthesis of the cationiclipids of Formula I as a racemic mixture or in optically pure form.

The terms “cationic lipid” and “amino lipid” are used interchangeablyherein to include those lipids and salts thereof having one, two, three,or more fatty acid or fatty alkyl chains and a pH-titratable amino headgroup (e.g., an alkylamino or dialkylamino head group). The cationiclipid is typically protonated (i.e., positively charged) at a pH belowthe pK_(a) of the cationic lipid and is substantially neutral at a pHabove the pK_(a). The cationic lipids of the invention may also betermed titratable cationic lipids.

The term “salts” includes any anionic and cationic complex, such as thecomplex formed between a cationic lipid disclosed herein and one or moreanions. Non-limiting examples of anions include inorganic and organicanions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate(e.g., hemioxalate), phosphate, phosphonate, hydrogen phosphate,dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite,nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate,hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate,lactate, acrylate, polyacrylate, fumarate, maleate, itaconate,glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate,polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite,bromate, hypobromite, iodate, an alkylsulfonate, an arylsulfonate,arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate,hydroxide, peroxide, permanganate, and mixtures thereof. In particularembodiments, the salts of the cationic lipids disclosed herein arecrystalline salts.

The term “alkyl” includes a straight chain or branched, noncyclic orcyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbonatoms. Representative saturated straight chain alkyls include, but arenot limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, andthe like, while saturated branched alkyls include, without limitation,isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like.Representative saturated cyclic alkyls include, but are not limited to,the C₃₋₈ cycloalkyls described herein, while unsaturated cyclic alkylsinclude, without limitation, the C₃₋₈ cycloalkenyls described herein.

The term “heteroalkyl,” includes a straight chain or branched, noncyclicor cyclic, saturated aliphatic hydrocarbon as defined above having fromabout 1 to about 5 heteroatoms (i.e., 1, 2, 3, 4, or 5 heteroatoms) suchas, for example, 0, N, Si, and/or S, wherein the nitrogen and sulfuratoms may optionally be oxidized and the nitrogen heteroatom mayoptionally be quaternized. The heteroalkyl group can be attached to theremainder of the molecule through a carbon atom or a heteroatom.

The term “cyclic alkyl” includes any of the substituted or unsubstitutedcycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenylgroups described below.

The term “cycloalkyl” includes a substituted or unsubstituted cyclicalkyl group having from about 3 to about 8 carbon atoms (i.e., 3, 4, 5,6, 7, or 8 carbon atoms) as ring vertices. Preferred cycloalkyl groupsinclude those having from about 3 to about 6 carbon atoms as ringvertices. Examples of C₃₋₈ cycloalkyl groups include, but are notlimited to, cyclopropyl, methyl-cyclopropyl, dimethyl-cyclopropyl,cyclobutyl, methyl-cyclobutyl, cyclopentyl, methyl-cyclopentyl,cyclohexyl, methyl-cyclohexyl, dimethyl-cyclohexyl, cycloheptyl, andcyclooctyl, as well as other substituted C₃₋₈ cycloalkyl groups.

The term “heterocycloalkyl” includes a substituted or unsubstitutedcyclic alkyl group as defined above having from about 1 to about 3heteroatoms as ring members selected from the group consisting of O, N,Si and S, wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheterocycloalkyl group can be attached to the remainder of the moleculethrough a carbon atom or a heteroatom.

The term “cycloalkenyl” includes a substituted or unsubstituted cyclicalkenyl group having from about 3 to about 8 carbon atoms (i.e., 3, 4,5, 6, 7, or 8 carbon atoms) as ring vertices. Preferred cycloalkenylgroups are those having from about 3 to about 6 carbon atoms as ringvertices. Examples of C₃₋₈ cycloalkenyl groups include, but are notlimited to, cyclopropenyl, methyl-cyclopropenyl, dimethyl-cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, andcyclooctenyl, as well as other substituted C₃₋₈ cycloalkenyl groups.

The term “heterocycloalkenyl” includes a substituted or unsubstitutedcyclic alkenyl group as defined above having from about 1 to about 3heteroatoms as ring members selected from the group consisting of O, N,Si and S, wherein the nitrogen and sulfur atoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Theheterocycloalkenyl group can be attached to the remainder of themolecule through a carbon atom or a heteroatom.

The term “alkoxy” includes a group of the formula alkyl-O—, wherein“alkyl” has the previously given definition. Non-limiting examples ofalkoxy groups include methoxy, ethoxy, n-propoxy, iso-propoxy, n-butoxy,iso-butoxy, sec-butoxy and tert-butoxy.

The term “alkenyl” includes an alkyl, as defined above, containing atleast one double bond between adjacent carbon atoms. Alkenyls includeboth cis and trans isomers. Representative straight chain and branchedalkenyls include, but are not limited to, ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike. Representative cyclic alkenyls are described above.

The term “alkynyl” includes any alkyl or alkenyl, as defined above,which additionally contains at least one triple bond between adjacentcarbons. Representative straight chain and branched alkynyls include,without limitation, acetylenyl, propynyl, 1-butynyl, 2-butynyl,1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

The term “aryl” includes a polyunsaturated, typically aromatic,hydrocarbon group which can be a single ring or multiple rings (up tothree rings) which are fused together or linked covalently, and whichoptionally carries one or more substituents, such as, for example,halogen, trifluoromethyl, amino, alkyl, alkoxy, alkylcarbonyl, cyano,carbamoyl, alkoxycarbamoyl, methylendioxy, carboxy, alkoxycarbonyl,aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, hydroxy, nitro,and the like. Non-limiting examples of unsubstituted aryl groups includephenyl, naphthyl, and biphenyl. Examples of substituted aryl groupsinclude, but are not limited to, phenyl, chlorophenyl,trifluoromethylphenyl, chlorofluorophenyl, and aminophenyl.

The terms “alkylthio,” “alkylsulfonyl,” “alkylsulfinyl,” and“arylsulfonyl” include groups having the formula —S—R^(i), —S(O)₂—R^(i),—S(O)—R^(i) and —S(O)₂R^(j), respectively, wherein R^(i) is an alkylgroup as previously defined and R^(j) is an aryl group as previouslydefined.

The terms “alkenyloxy” and “alkynyloxy” include groups having theformula —O—R^(i), wherein R^(i) is an alkenyl or alkynyl group,respectively.

The terms “alkenylthio” and “alkynylthio” include groups having theformula —S—R^(k), wherein R^(k) is an alkenyl or alkynyl group,respectively.

The term “alkoxycarbonyl” includes a group having the formula—C(O)O—R^(i), wherein R^(i) is an alkyl group as defined above andwherein the total number of carbon atoms refers to the combined alkyland carbonyl moieties.

The term “acyl” includes any alkyl, alkenyl, or alkynyl wherein thecarbon at the point of attachment is substituted with an oxo group, asdefined below. The following are non-limiting examples of acyl groups:—C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl.

The term “heterocycle” includes a 5- to 7-membered monocyclic, or 7- to10-membered bicyclic, heterocyclic ring which is either saturated,unsaturated, or aromatic, and which contains from 1 or 2 heteroatomsindependently selected from nitrogen, oxygen and sulfur, and wherein thenitrogen and sulfur heteroatoms may be optionally oxidized, and thenitrogen heteroatom may be optionally quaternized, including bicyclicrings in which any of the above heterocycles are fused to a benzenering. The heterocycle may be attached via any heteroatom or carbon atom.Heterocycles include, but are not limited to, heteroaryls as definedbelow, as well as morpholinyl, pyrrolidinonyl, pyrrolidinyl,piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl,oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl,tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, andthe like.

The term “heteroaryl” includes an aromatic 5- to 10-membered heterocyclewhich contains one, two, or more heteroatoms selected from nitrogen (N),oxygen (O), and sulfur (S). The heteroaryl can be substituted on one ormore carbon atoms with substituents such as, for example, halogen,alkyl, alkoxy, cyano, haloalkyl (e.g., trifluoromethyl), heterocyclyl(e.g., morpholinyl or pyrrolidinyl), and the like. Non-limiting examplesof heteroaryls include pyridinyl and furanyl.

The term “halogen” includes fluoro, chloro, bromo, and iodo.

The terms “optionally substituted alkyl,” “optionally substituted cyclicalkyl,” “optionally substituted alkenyl,” “optionally substitutedalkynyl,” “optionally substituted acyl,” and “optionally substitutedheterocycle” mean that, when substituted, at least one hydrogen atom isreplaced with a substituent. In the case of an “oxo” substituent (═O),two hydrogen atoms are replaced. Non-limiting examples of substituentsinclude oxo, halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0,1, or 2, R^(x) and R^(y) are the same or different and are independentlyhydrogen, alkyl, or heterocycle, and each of the alkyl and heterocyclesubstituents may be further substituted with one or more of oxo,halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x), and —SO_(n)NR^(x)R^(y). The term“optionally substituted,” when used before a list of substituents, meansthat each of the substituents in the list may be optionally substitutedas described herein.

In one aspect, the present invention provides a cationic lipid ofFormula I having the following structure:

or salts thereof, wherein:

R¹ and R² are either the same or different and are independentlyhydrogen (H) or an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, orC₂-C₆ alkynyl, or R¹ and R² may join to form an optionally substitutedheterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selectedfrom the group consisting of nitrogen (N), oxygen (O), and mixturesthereof;

R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to provide aquaternary amine;

R⁴ and R⁵ are either the same or different and are independently anoptionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl, C₁₀-C₂₄ alkynyl,or C₁₀-C₂₄ acyl;

X is O, S, N(R⁶), C(O), C(O)O, OC(O), C(O)N(R⁶), N(R⁶)C(O), OC(O)N(R⁶),N(R⁶)C(O)O, C(O)S, C(S)O, S(O), S(O)(O), or C(S), wherein R⁶ is hydrogen(H) or an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆alkynyl; and

Y is either absent or is an optionally substituted C₁-C₆ alkyl, C₂-C₆alkenyl, or C₂-C₆ alkynyl.

In some embodiments, R¹, R², and R⁶ are each independently hydrogen (H)or an optionally substituted C₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl,C₁-C₅ alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅ alkyl, C₂-C₆ alkyl, C₃-C₄alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl, C₄-C₅ alkyl, C₄-C₆ alkyl, C₅-C₆ alkyl,C₂-C₃ alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl, C₂-C₆ alkenyl, C₃-C₄alkenyl, C₃-C₅ alkenyl, C₃-C₆ alkenyl, C₄-C₅ alkenyl, C₄-C₆ alkenyl,C₅-C₆ alkenyl, C₂-C₃ alkynyl, C₂-C₄ alkynyl, C₂-C₅ alkynyl, C₂-C₆alkynyl, C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆ alkynyl, C₄-C₅ alkynyl,C₄-C₆ alkynyl, or C₅-C₆ alkynyl. In other embodiments, R¹ and R² arejoined to form a heterocyclic ring of 5 carbon atoms and 1 nitrogenatom, wherein the heterocyclic ring can be substituted with asubstituent such as a hydroxyl (—OH) group at the ortho, meta, and/orpara positions. In particular embodiments, R¹ and R² are both methylgroups. In certain instances, R³ is absent when the pH is above thepK_(a) of the cationic lipid and R³ is hydrogen (H) when the pH is belowthe pK_(a) of the cationic lipid such that the amino head group isprotonated. In certain other instances, R³ is an optionally substitutedC₁-C₂ alkyl, C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₂-C₃ alkyl, C₂-C₄alkyl, C₂-C₅ alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl,C₄-C₅ alkyl, C₄-C₆ alkyl, or C₅-C₆ alkyl to provide a quaternary amine.In one particular embodiment, X is C(O)O. In another particularembodiment, X is O. In certain other embodiments, X is C(O)N(R⁶),N(R⁶)C(O)O, or C(O)S. In one particular embodiment, X is N(R⁶)C(O)O andR⁶ is hydrogen (H) or a methyl group. In other instances, R¹ and R² arenot both methyl groups when X is C(O)O, Y is (CH₂)₂ or (CH₂)₃, and R⁴and R⁵ are both linoleyl moieties.

In certain embodiments, at least one or both R⁴ and R⁵ independentlycomprises an optionally substituted C₁₂-C₂₄, C₁₂-C₂₂, C₁₂-C₂₀, C₁₄-C₂₄,C₁₄-C₂₂, C₁₄-C₂₀, C₁₆-C₂₄, C₁₆-C₂₂, or C₁₆-C₂₀ alkyl or acyl group(i.e., C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, orC₂₄ alkyl or acyl group). In other embodiments, at least one or both R⁴and R⁵ independently comprises at least 1, 2, 3, 4, 5, or 6 sites ofunsaturation (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6 sitesof unsaturation) or a substituted alkyl or acyl group. In certaininstances, the the unsaturated side-chain may comprise a myristoleylmoiety, a palmitoleyl moiety, an oleyl moiety, a dodecadienyl moiety, atetradecadienyl moiety, a hexadecadienyl moiety, an octadecadienylmoiety, an icosadienyl moiety, a dodecatrienyl moiety, a tetradectrienylmoiety, a hexadecatrienyl moiety, an octadecatrienyl moiety, anicosatrienyl moiety, or an acyl derivative thereof (e.g., linoleoyl,linolenoyl, γ-linolenoyl, etc.). In some instances, the octadecadienylmoiety is a linoleyl moiety. In particular embodiments, R⁴ and R⁵ areboth linoleyl moieties. In other instances, the octadecatrienyl moietyis a linolenyl moiety or a γ-linolenyl moiety. In particularembodiments, R⁴ and R⁵ are both linolenyl moieties or γ-linolenylmoieties. In embodiments where one or both R⁴ and R⁵ independentlycomprises a branched alkyl or acyl group (e.g., a substituted alkyl oracyl group), the branched alkyl or acyl group may comprise a C₁₂-C₂₄alkyl or acyl having at least 1-6 (e.g., 1, 2, 3, 4, 5, 6, or more)C₁-C₆ alkyl substituents. In particular embodiments, the branched alkylor acyl group comprises a C₁₂-C₂₀ or C₁₄-C₂₂ alkyl or acyl with 1-6(e.g., 1, 2, 3, 4, 5, 6) C₁-C₄ alkyl (e.g., methyl, ethyl, propyl, orbutyl) substituents. In some embodiments, the branched alkyl groupcomprises a phytanyl (3,7,11,15-tetramethyl-hexadecanyl) moiety and thebranched acyl group comprises a phytanoyl(3,7,11,15-tetramethyl-hexadecanoyl) moiety. In particular embodiments,R⁴ and R⁵ are both phytanyl moieties.

In other embodiments, at least one or both R⁴ and R⁵ independentlycomprises at least 1, 2, 3, 4, 5, 6, or more optionally substitutedcyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or2-6 optionally substituted cyclic alkyl groups). In certain instances,at least one or both R⁴ and R⁵ independently comprises an optionallysubstituted C₁₂-C₂₄, C₁₂-C₂₂, C₁₂-C₂₀, C₁₄-C₂₄, C₁₄-C₂₂, C₁₄-C₂₀,C₁₆-C₂₄, C₁₆-C₂₂, or C₁₆-C₂₀ alkyl, alkenyl, alkynyl, or acyl group(i.e., C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, orC₂₄ alkyl, alkenyl, alkynyl, or acyl group), wherein at least one of R⁴and R⁵ comprises at least 1, 2, 3, 4, 5, or 6 optionally substitutedcyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or2-6 optionally substituted cyclic alkyl groups).

In particular embodiments, one or more of the optionally substitutedcyclic alkyl groups present in R⁴ and/or R⁵ are independently selectedfrom the group consisting of an optionally substituted saturated cyclicalkyl group, an optionally substituted unsaturated cyclic alkyl group,and combinations thereof. In certain instances, the optionallysubstituted saturated cyclic alkyl group comprises an optionallysubstituted C₃₋₈ cycloalkyl group (e.g., cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.). In preferredembodiments, the optionally substituted saturated cyclic alkyl groupcomprises a cyclopropyl group, optionally containing one or moresubstituents and/or heteroatoms. In other instances, the optionallysubstituted unsaturated cyclic alkyl group comprises an optionallysubstituted C₃₋₈ cycloalkenyl group (e.g., cyclopropenyl, cyclobutenyl,cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, etc.).

In some embodiments, one of R⁴ or R⁵ comprises at least 1, 2, 3, 4, 5,or 6 sites of unsaturation (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4,2-5, or 2-6 sites of unsaturation) or a substituted alkyl or acyl group,and the other side-chain comprises at least 1, 2, 3, 4, 5, or 6optionally substituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5,1-6, 2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkyl groups).In embodiments where one of R⁴ or R⁵ comprises at least 1, 2, 3, 4, 5,or 6 sites of unsaturation, the unsaturated side-chain may comprise amyristoleyl moiety, a palmitoleyl moiety, an oleyl moiety, adodecadienyl moiety, a tetradecadienyl moiety, a hexadecadienyl moiety,an octadecadienyl moiety, an icosadienyl moiety, a dodecatrienyl moiety,a tetradectrienyl moiety, a hexadecatrienyl moiety, an octadecatrienylmoiety, an icosatrienyl moiety, or an acyl derivative thereof (e.g.,linoleoyl, linolenoyl, γ-linolenoyl, etc.). In some instances, theoctadecadienyl moiety is a linoleyl moiety. In other instances, theoctadecatrienyl moiety is a linolenyl moiety or a γ-linolenyl moiety. Inembodiments where one of R⁴ or R⁵ comprises a branched alkyl or acylgroup (e.g., a substituted alkyl or acyl group), the branched alkyl oracyl group may comprise a C₁₂-C₂₄ alkyl or acyl having at least 1-6(e.g., 1, 2, 3, 4, 5, 6, or more) C₁-C₆ alkyl substituents. Inparticular embodiments, the branched alkyl or acyl group comprises aC₁₂-C₂₀ or C₁₄-C₂₂ alkyl or acyl with 1-6 (e.g., 1, 2, 3, 4, 5, 6) C₁-C₄alkyl (e.g., methyl, ethyl, propyl, or butyl) substituents. In someembodiments, the branched alkyl group comprises a phytanyl moiety andthe branched acyl group comprises a phytanoyl moiety.

In particular embodiments, R⁴ and R⁵ are both independently selectedC₁₂-C₂₀ alkyl groups (i.e., C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, orC₂₀ alkyl groups) having at least 1, 2, 3, 4, 5, or 6 optionallysubstituted cyclic alkyl groups (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3,2-4, 2-5, or 2-6 optionally substituted cyclic alkyl groups). Inpreferred embodiments, R⁴ and R⁵ are both C₁₈ alkyl groups having atleast one, two, three, or more optionally substituted cyclic alkylgroups such as, for example, an optionally substituted C₃₋₈ cycloalkylgroup (e.g., a cyclopropyl group, optionally containing one or moresubstituents and/or heteroatoms). In certain embodiments, each of theoptionally substituted cyclic alkyl groups is independently selected andcan be the same cyclic alkyl group (e.g., all cyclopropyl groups) ordifferent cyclic alkyl groups (e.g., cyclopropyl and other cycloalkyl,heterocycloalkyl, cycloalkenyl, and/or heterocycloalkenyl groups).

In preferred embodiments, the optionally substituted cyclic alkyl groupspresent in R⁴ and/or R⁵ are located at the site(s) of unsaturation inthe corresponding unsaturated side-chain. As a non-limiting example, oneor both of R⁴ and R⁵ are C₁₈ alkyl groups having 1, 2, or 3 optionallysubstituted cyclic alkyl groups, wherein the optionally substitutedcyclic alkyl groups (e.g., independently selected cyclopropyl groups)are located at one or more (e.g., all) of the sites of unsaturationpresent in a corresponding linoleyl moiety, linolenyl moiety, orγ-linolenyl moiety.

In alternative embodiments to the cationic lipid of Formula I, R⁴ and R⁵are different and are independently an optionally substituted C₁-C₂₄alkyl, C₂-C₂₄ alkenyl, C₂′ C₂₄ alkynyl, or C₁-C₂₄ acyl. In particularembodiments, at least one or both R⁴ and R⁵ comprises at least 1, 2, 3,4, 5, or 6 optionally substituted cyclic alkyl groups (e.g., 1-2, 1-3,1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6 optionally substituted cyclic alkylgroups). In certain embodiments, R⁴ and R⁵ are different and areindependently an optionally substituted C₄-C₂₀ alkyl, C₄-C₂₀ alkenyl,C₄-C₂₀ alkynyl, or C₄-C₂₀ acyl. In some instances, R⁴ is an optionallysubstituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄acyl, and R⁵ is an optionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl,C₄-C₁₀ alkynyl, or C₄-C₁₀ acyl. In other instances, R⁴ is an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl, and R⁵ is anoptionally substituted C₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl, C₄-C₈ orC₆ alkynyl, or C₄-C₈ or C₆ acyl. In certain instances, R⁴ is anoptionally substituted C₄-C₁₀ alkyl, C₄-C₁₀ alkenyl, alkynyl, or C₄-C₁₀acyl, and R⁵ is an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl. In certain other instances,R⁴ is an optionally substituted C₄-C₈ or C₆ alkyl, C₄-C₈ or C₆ alkenyl,C₄-C₈ or C₆ alkynyl, or C₄-C₈ or C₆ acyl, and R⁵ is an optionallysubstituted C₁₂-C₂₀ or C₁₄-C₂₂ alkyl, C₁₂-C₂₀ or C₁₄-C₂₂ alkenyl,C₁₂-C₂₀ or C₁₄-C₂₂ alkynyl, or C₁₂-C₂₀ or C₁₄-C₂₂ acyl. In particularembodiments, one or more of the optionally substituted cyclic alkylgroups, when present in R⁴ and/or R⁵, are as described above.

In some groups of embodiments to the cationic lipid of Formula I, R⁴ andR⁵ are either the same or different and are independently selected fromthe group consisting of:

In certain embodiments, Y is an optionally substituted C₁-C₂ alkyl,C₁-C₃ alkyl, C₁-C₄ alkyl, C₁-C₅ alkyl, C₂-C₃ alkyl, C₂-C₄ alkyl, C₂-C₅alkyl, C₂-C₆ alkyl, C₃-C₄ alkyl, C₃-C₅ alkyl, C₃-C₆ alkyl, C₄-C₅ alkyl,C₄-C₆ alkyl, C₅-C₆ alkyl, C₂-C₃ alkenyl, C₂-C₄ alkenyl, C₂-C₅ alkenyl,C₂-C₆ alkenyl, C₃-C₄ alkenyl, C₃-C₅ alkenyl, C₃-C₆ alkenyl, C₄-C₅alkenyl, C₄-C₆ alkenyl, C₅-C₆ alkenyl, C₂-C₃ alkynyl, C₂-C₄ alkynyl,C₂-C₅ alkynyl, C₂-C₆ alkynyl, C₃-C₄ alkynyl, C₃-C₅ alkynyl, C₃-C₆alkynyl, C₄-C₅ alkynyl, C₄-C₆ alkynyl, or C₅-C₆ alkynyl. In oneparticular embodiment, Y is (CH₂)_(n) and n is 0, 1, 2, 3, 4, 5, or 6(e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 2-3, 2-4, 2-5, or 2-6). In a preferredembodiment, n is 2, 3, or 4.

In particular embodiments, the cationic lipid of Formula I has thefollowing structure:

or salts thereof, wherein R¹, R², R³, R⁴, R⁵, X, and n are the same asdescribed above.

In some embodiments, the cationic lipid of Formula I forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the cationic lipid of Formula I is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

In particularly preferred embodiments, the cationic lipid of Formula Ihas one of the following structures:

The compounds of the invention may be prepared by known organicsynthesis techniques, including the methods described in the Examples.In some embodiments, the synthesis of the cationic lipids of theinvention may require the use of protecting groups. Protecting groupmethodology is well known to those skilled in the art (see, e.g.,PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, Green, T. W. et. al.,Wiley-Interscience, New York City, 1999). Briefly, protecting groupswithin the context of this invention are any group that reduces oreliminates the unwanted reactivity of a functional group. A protectinggroup can be added to a functional group to mask its reactivity duringcertain reactions and then removed to reveal the original functionalgroup. In certain instances, an “alcohol protecting group” is used. An“alcohol protecting group” is any group which decreases or eliminatesthe unwanted reactivity of an alcohol functional group. Protectinggroups can be added and removed using techniques well known in the art.

In certain embodiments, the cationic lipids of the present inventionhave at least one protonatable or deprotonatable group, such that thelipid is positively charged at a pH at or below physiological pH (e.g.,pH 7.4), and neutral at a second pH, preferably at or abovephysiological pH. It will be understood by one of ordinary skill in theart that the addition or removal of protons as a function of pH is anequilibrium process, and that the reference to a charged or a neutrallipid refers to the nature of the predominant species and does notrequire that all of the lipid be present in the charged or neutral form.Lipids that have more than one protonatable or deprotonatable group, orwhich are zwiterrionic, are not excluded from use in the invention.

In certain other embodiments, protonatable lipids according to theinvention have a pK_(a) of the protonatable group in the range of about4 to about 11. Most preferred is a pK_(a) of about 4 to about 7, becausethese lipids will be cationic at a lower pH formulation stage, whileparticles will be largely (though not completely) surface neutralized atphysiological pH of around pH 7.4. One of the benefits of this pK_(a) isthat at least some nucleic acid associated with the outside surface ofthe particle will lose its electrostatic interaction at physiological pHand be removed by simple dialysis, thus greatly reducing the particle'ssusceptibility to clearance.

2. Other Cationic Lipids

Other cationic lipids or salts thereof which may also be included in thelipid particles of the present invention include, but are not limitedto, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA),1,2-dilinoleyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDMA),1,2-dilinoleoyloxy-(N,N-dimethyl)-butyl-4-amine (C2-DLinDAP),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA;also known as “XTC2” or “C2K”),2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA;“C3K”), 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane(DLin-K-C4-DMA; “C4K”),2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA),2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ),2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DO-K-DMA),2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DS-K-DMA),2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane (DLin-K-MA),2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride(DLin-K-TMA.Cl),2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane(DLin-K²-DMA), 2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane(D-Lin-K-N-methylpiperzine),(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-M-C3-DMA; “MC3”),dilinoleylmethyl-3-dimethylaminopropionate (DLin-M-C2-DMA; also known asDLin-M-K-DMA or DLin-M-DMA),1,2-dioeylcarbamoyloxy-3-dimethylaminopropane (DO-C-DAP),1,2-dimyristoleoyl-3-dimethylaminopropane (DMDAP),1,2-dioleoyl-3-trimethylaminopropane chloride (DOTAP.Cl),1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanedio (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane(CLinDMA),2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethy-1-(cis,cis-9′,1-2′-octadecadienoxy)propane(CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA),1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP),1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP),N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA),1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE),2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyll-N,N-dimethyl-1-propanaminiumtrifluoroacetate(DOSPA), dioctadecylamidoglycyl spermine (DOGS), and mixtures thereof.

Additional cationic lipids or salts thereof which may be present in thelipid particles described herein include novel cyclic cationic lipidssuch as CP-LenMC3, CP-γ-LenMC3, CP-MC3, CP-DLen-C2K-DMA,CP-γDLen-C2K-DMA, CP-C2K-DMA, CP-DODMA, CP-DPetroDMA, CP-DLinDMA,CP-DLenDMA, CP-γDLenDMA, analogs thereof, and combinations thereof, asdescribed in U.S. Provisional Application No. 61/334,096 entitled “NovelCyclic Cationic Lipids and Methods of Use Thereof,” bearing AttorneyDocket No. 020801-010100US, filed May 12, 2010. Additional cationiclipids or salts thereof which may be present in the lipid particlesdescribed herein include novel cationic lipids such as 4-B10, 4-B12,4-B13, 4-B14, 4-B15, 4-B16, γ-4-B10,N,N-dimethyl-2-((11Z,14Z)-3-((9Z,12Z)-octadeca-9,12-dienyloxy)icosa-11,14-dienyloxy)ethanamine(4-B10 Ether), (11Z,14Z)-3-(dimethylamino)propyl3-((9Z,12Z)-octadeca-9,12-dienoyloxy)icosa-11,14-dienoate, CP-4-B10,analogs thereof, and combinations thereof, as described in U.S.Provisional Application No. 61/334,087 entitled “Novel Cationic Lipidsand Methods of Use Thereof,” bearing Attorney Docket No.020801-010800US, filed May 12, 2010. Additional cationic lipids or saltsthereof which may be present in the lipid particles described hereininclude the novel cationic lipids described in U.S. ProvisionalApplication No. 61/295,134, entitled “Improved Cationic Lipids andMethods for the Delivery of Nucleic Acids,” filed Jan. 14, 2010.Additional cationic lipids or salts thereof which may be present in thelipid particles described herein include the cationic lipids describedin U.S. Patent Publication No. 20090023673. The disclosures of each ofthese patent documents are herein incorporated by reference in theirentirety for all purposes.

In some embodiments, the additional cationic lipid forms a salt(preferably a crystalline salt) with one or more anions. In oneparticular embodiment, the additional cationic lipid is the oxalate(e.g., hemioxalate) salt thereof, which is preferably a crystallinesalt.

The synthesis of cationic lipids such as DLinDMA and DLenDMA, as well asadditional cationic lipids, is described in U.S. Patent Publication No.20060083780, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of cationic lipids such as γ-DLenDMA, C2-DLinDMA andC2-DLinDAP, as well as additional cationic lipids, is described in U.S.Provisional Application No. 61/295,134, entitled “Improved CationicLipids and Methods for the Delivery of Nucleic Acids,” filed Jan. 14,2010, the disclosure of which is herein incorporated by reference in itsentirety for all purposes.

The synthesis of cationic lipids such as DLin-K-DMA, as well asadditional cationic lipids, is described in PCT Publication No. WO09/086558, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of cationic lipids such as DLin-K-C2-DMA, DLin-K-C3-DMA,DLin-K-C4-DMA, DLin-K6-DMA, DLin-K-MPZ, DO-K-DMA, DS-K-DMA, DLin-K-MA,DLin-K-TMA.Cl, DLin-K²-DMA, D-Lin-K-N-methylpiperzine, DLin-M-C2-DMA,DO-C-DAP, DMDAP, and DOTAP.Cl, as well as additional cationic lipids, isdescribed in PCT Publication No. WO 2010/042877, entitled “ImprovedAmino Lipids and Methods for the Delivery of Nucleic Acids,” filed Oct.9, 2009, the disclosure of which is incorporated herein by reference inits entirety for all purposes.

The synthesis of DLin-M-C3-DMA (“MC3”), as well as additional cationiclipids (e.g., certain analogs of MC3), is described herein and in U.S.Provisional Application No. 61/185,800, entitled “Novel Lipids andCompositions for the Delivery of Therapeutics,” filed Jun. 10, 2009, andU.S. Provisional Application No. 61/287,995, entitled “Methods andCompositions for Delivery of Nucleic Acids,” filed Dec. 18, 2009, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes.

The synthesis of cationic lipids such as DLin-C-DAP, DLinDAC, DLinMA,DLinDAP, DLin-S-DMA, DLin-2-DMAP, DLinTMA.Cl, DLinTAP.Cl, DLinMPZ,DLinAP, DOAP, and DLin-EG-DMA, as well as additional cationic lipids, isdescribed in PCT Publication No. WO 09/086558, the disclosure of whichis herein incorporated by reference in its entirety for all purposes.

The synthesis of cationic lipids such as CLinDMA, as well as additionalcationic lipids, is described in U.S. Patent Publication No.20060240554, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The synthesis of a number of other cationic lipids and related analogshas been described in U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833;5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO96/10390, the disclosures of which are each herein incorporated byreference in their entirety for all purposes. Additionally, a number ofcommercial preparations of cationic lipids can be used, such as, e.g.,LIPOFECTIN® (including DOTMA and DOPE, available from GIBCO/BRL);LIPOFECTAMINE® (including DOSPA and DOPE, available from GIBCO/BRL); andTRANSFECTAM® (including DOGS, available from Promega Corp.).

In some embodiments, the cationic lipid comprises from about 50 mol % toabout 90 mol %, from about 50 mol % to about 85 mol %, from about 50 mol% to about 80 mol %, from about 50 mol % to about 75 mol %, from about50 mol % to about 70 mol %, from about 50 mol % to about 65 mol %, fromabout 50 mol % to about 60 mol %, from about 55 mol % to about 65 mol %,or from about 55 mol % to about 70 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle. In particularembodiments, the cationic lipid comprises about 50 mol %, 51 mol %, 52mol %, 53 mol %, 54 mol %, 55 mol %, 56 mol %, 57 mol %, 58 mol %, 59mol %, 60 mol %, 61 mol %, 62 mol %, 63 mol %, 64 mol %, or 65 mol % (orany fraction thereof) of the total lipid present in the particle.

In other embodiments, the cationic lipid comprises from about 2 mol % toabout 60 mol %, from about 5 mol % to about 50 mol %, from about 10 mol% to about 50 mol %, from about 20 mol % to about 50 mol %, from about20 mol % to about 40 mol %, from about 30 mol % to about 40 mol %, orabout 40 mol % (or any fraction thereof or range therein) of the totallipid present in the particle.

Additional percentages and ranges of cationic lipids suitable for use inthe lipid particles of the present invention are described in PCTPublication No. WO 09/127060, U.S. Provisional Application No.61/184,652, filed Jun. 5, 2009, U.S. Provisional Application No.61/295,134, filed Jan. 14, 2010, and U.S. Provisional Application No.61/222,469, filed Jul. 1, 2009, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of cationic lipid present inthe lipid particles of the invention is a target amount, and that theactual amount of cationic lipid present in the formulation may vary, forexample, by ±5 mol %. For example, in the 1:57 lipid particle (e.g.,SNALP) formulation, the target amount of cationic lipid is 57.1 mol %,but the actual amount of cationic lipid may be ±5 mol %, ±4 mol %, ±3mol %, ±2 mol %, ±1 mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1mol % of that target amount, with the balance of the formulation beingmade up of other lipid components (adding up to 100 mol % of totallipids present in the particle).

B. Non-Cationic Lipids

The non-cationic lipids used in the lipid particles of the invention(e.g., SNALP) can be any of a variety of neutral uncharged,zwitterionic, or anionic lipids capable of producing a stable complex.

Non-limiting examples of non-cationic lipids include phospholipids suchas lecithin, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin,phosphatidic acid, cerebrosides, dicetylphosphate,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoyl-phosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),palmitoyloleyol-phosphatidylglycerol (POPG),dioleoylphosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),dipalmitoyl-phosphatidylethanolamine (DPPE),dimyristoyl-phosphatidylethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,dielaidoyl-phosphatidylethanolamine (DEPE),stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine,dilinoleoylphosphatidylcholine, and mixtures thereof. Otherdiacylphosphatidylcholine and diacylphosphatidylethanolaminephospholipids can also be used. The acyl groups in these lipids arepreferably acyl groups derived from fatty acids having C₁₀-C₂₄ carbonchains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

Additional examples of non-cationic lipids include sterols such ascholesterol and derivatives thereof. Non-limiting examples ofcholesterol derivatives include polar analogues such as 5α-cholestanol,5β-coprostanol, cholesteryl-(2′-hydroxy)-ethyl ether,cholesteryl-(4′-hydroxy)-butyl ether, and 6-ketocholestanol; non-polaranalogues such as 5α-cholestane, cholestenone, 5α-cholestanone,5β-cholestanone, and cholesteryl decanoate; and mixtures thereof. Inpreferred embodiments, the cholesterol derivative is a polar analoguesuch as cholesteryl-(4′-hydroxy)-butyl ether. The synthesis ofcholesteryl-(2′-hydroxy)-ethyl ether is described in PCT Publication No.WO 09/127060, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

In some embodiments, the non-cationic lipid present in the lipidparticles (e.g., SNALP) comprises or consists of a mixture of one ormore phospholipids and cholesterol or a derivative thereof. In otherembodiments, the non-cationic lipid present in the lipid particles(e.g., SNALP) comprises or consists of one or more phospholipids, e.g.,a cholesterol-free lipid particle formulation. In yet other embodiments,the non-cationic lipid present in the lipid particles (e.g., SNALP)comprises or consists of cholesterol or a derivative thereof, e.g., aphospholipid-free lipid particle formulation.

Other examples of non-cationic lipids suitable for use in the presentinvention include nonphosphorous containing lipids such as, e.g.,stearylamine, dodecylamine, hexadecylamine, acetyl palmitate,glycerolricinoleate, hexadecyl stereate, isopropyl myristate, amphotericacrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfatepolyethyloxylated fatty acid amides, dioctadecyldimethyl ammoniumbromide, ceramide, sphingomyelin, and the like.

In some embodiments, the non-cationic lipid comprises from about 10 mol% to about 60 mol %, from about 20 mol % to about 55 mol %, from about20 mol % to about 45 mol %, from about 20 mol % to about 40 mol %, fromabout 25 mol % to about 50 mol %, from about 25 mol % to about 45 mol %,from about 30 mol % to about 50 mol %, from about 30 mol % to about 45mol %, from about 30 mol % to about 40 mol %, from about 35 mol % toabout 45 mol %, from about 37 mol % to about 42 mol %, or about 35 mol%, 36 mol %, 37 mol %, 38 mol %, 39 mol %, 40 mol %, 41 mol %, 42 mol %,43 mol %, 44 mol %, or 45 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle.

In embodiments where the lipid particles contain a mixture ofphospholipid and cholesterol or a cholesterol derivative, the mixturemay comprise up to about 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60mol % of the total lipid present in the particle.

In some embodiments, the phospholipid component in the mixture maycomprise from about 2 mol % to about 20 mol %, from about 2 mol % toabout 15 mol %, from about 2 mol % to about 12 mol %, from about 4 mol %to about 15 mol %, or from about 4 mol % to about 10 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the phospholipid componentin the mixture comprises from about 5 mol % to about 10 mol %, fromabout 5 mol % to about 9 mol %, from about 5 mol % to about 8 mol %,from about 6 mol % to about 9 mol %, from about 6 mol % to about 8 mol%, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a 1:57 lipid particle formulationcomprising a mixture of phospholipid and cholesterol may comprise aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof), e.g., in a mixture with cholesterol or a cholesterolderivative at about 34 mol % (or any fraction thereof) of the totallipid present in the particle. As another non-limiting example, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise a phospholipid such as DPPC or DSPC at about 7mol % (or any fraction thereof), e.g., in a mixture with cholesterol ora cholesterol derivative at about 32 mol % (or any fraction thereof) ofthe total lipid present in the particle.

In other embodiments, the cholesterol component in the mixture maycomprise from about 25 mol % to about 45 mol %, from about 25 mol % toabout 40 mol %, from about 30 mol % to about 45 mol %, from about 30 mol% to about 40 mol %, from about 27 mol % to about 37 mol %, from about25 mol % to about 30 mol %, or from about 35 mol % to about 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. In certain preferred embodiments, the cholesterol component inthe mixture comprises from about 25 mol % to about 35 mol %, from about27 mol % to about 35 mol %, from about 29 mol % to about 35 mol %, fromabout 30 mol % to about 35 mol %, from about 30 mol % to about 34 mol %,from about 31 mol % to about 33 mol %, or about 30 mol %, 31 mol %, 32mol %, 33 mol %, 34 mol %, or 35 mol % (or any fraction thereof or rangetherein) of the total lipid present in the particle. Typically, a 1:57lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise cholesterol or a cholesterol derivative atabout 34 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle. Typically, a 7:54lipid particle formulation comprising a mixture of phospholipid andcholesterol may comprise cholesterol or a cholesterol derivative atabout 32 mol % (or any fraction thereof), e.g., in a mixture with aphospholipid such as DPPC or DSPC at about 7 mol % (or any fractionthereof) of the total lipid present in the particle.

In embodiments where the lipid particles are phospholipid-free, thecholesterol or derivative thereof may comprise up to about 25 mol %, 30mol %, 35 mol %, 40 mol %, 45 mol %, 50 mol %, 55 mol %, or 60 mol % ofthe total lipid present in the particle.

In some embodiments, the cholesterol or derivative thereof in thephospholipid-free lipid particle formulation may comprise from about 25mol % to about 45 mol %, from about 25 mol % to about 40 mol %, fromabout 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %,from about 31 mol % to about 39 mol %, from about 32 mol % to about 38mol %, from about 33 mol % to about 37 mol %, from about 35 mol % toabout 45 mol %, from about 30 mol % to about 35 mol %, from about 35 mol% to about 40 mol %, or about 30 mol %, 31 mol %, 32 mol %, 33 mol %, 34mol %, 35 mol %, 36 mol %, 37 mol %, 38 mol %, 39 mol %, or 40 mol % (orany fraction thereof or range therein) of the total lipid present in theparticle. As a non-limiting example, a 1:62 lipid particle formulationmay comprise cholesterol at about 37 mol % (or any fraction thereof) ofthe total lipid present in the particle. As another non-limitingexample, a 7:58 lipid particle formulation may comprise cholesterol atabout 35 mol % (or any fraction thereof) of the total lipid present inthe particle.

In other embodiments, the non-cationic lipid comprises from about 5 mol% to about 90 mol %, from about 10 mol % to about 85 mol %, from about20 mol % to about 80 mol %, about 10 mol % (e.g., phospholipid only), orabout 60 mol % (e.g., phospholipid and cholesterol or derivativethereof) (or any fraction thereof or range therein) of the total lipidpresent in the particle.

Additional percentages and ranges of non-cationic lipids suitable foruse in the lipid particles of the present invention are described in PCTPublication No. WO 09/127060, U.S. Provisional Application No.61/184,652, filed Jun. 5, 2009, U.S. Provisional Application No.61/295,134, filed Jan. 14, 2010, and U.S. Provisional Application No.61/222,469, filed Jul. 1, 2009, the disclosures of which are hereinincorporated by reference in their entirety for all purposes.

It should be understood that the percentage of non-cationic lipidpresent in the lipid particles of the invention is a target amount, andthat the actual amount of non-cationic lipid present in the formulationmay vary, for example, by ±5 mol %. For example, in the 1:57 lipidparticle (e.g., SNALP) formulation, the target amount of phospholipid is7.1 mol % and the target amount of cholesterol is 34.3 mol %, but theactual amount of phospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %,±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that targetamount, and the actual amount of cholesterol may be ±3 mol %, 2 mol %, 1mol %, 0.75 mol %, 0.5 mol %, 0.25 mol %, or ±0.1 mol % of that targetamount, with the balance of the formulation being made up of other lipidcomponents (adding up to 100 mol % of total lipids present in theparticle). Similarly, in the 7:54 lipid particle (e.g., SNALP)formulation, the target amount of phospholipid is 6.75 mol % and thetarget amount of cholesterol is 32.43 mol %, but the actual amount ofphospholipid may be ±2 mol %, ±1.5 mol %, ±1 mol %, ±0.75 mol %, ±0.5mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, and the actualamount of cholesterol may be ±3 mol %, ±2 mol %, ±1 mol %, ±0.75 mol %,±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of that target amount, with thebalance of the formulation being made up of other lipid components(adding up to 100 mol % of total lipids present in the particle).

C. Lipid Conjugates

In addition to cationic and non-cationic lipids, the lipid particles ofthe invention (e.g., SNALP) may further comprise a lipid conjugate. Theconjugated lipid is useful in that it prevents the aggregation ofparticles. Suitable conjugated lipids include, but are not limited to,PEG-lipid conjugates, POZ-lipid conjugates, ATTA-lipid conjugates,cationic-polymer-lipid conjugates (CPLs), and mixtures thereof. Incertain embodiments, the particles comprise either a PEG-lipid conjugateor an ATTA-lipid conjugate together with a CPL.

In a preferred embodiment, the lipid conjugate is a PEG-lipid. Examplesof PEG-lipids include, but are not limited to, PEG coupled todialkyloxypropyls (PEG-DAA) as described in, e.g., PCT Publication No.WO 05/026372, PEG coupled to diacylglycerol (PEG-DAG) as described in,e.g., U.S. Patent Publication Nos. 20030077829 and 2005008689, PEGcoupled to phospholipids such as phosphatidylethanolamine (PEG-PE), PEGconjugated to ceramides as described in, e.g., U.S. Pat. No. 5,885,613,PEG conjugated to cholesterol or a derivative thereof, and mixturesthereof. The disclosures of these patent documents are hereinincorporated by reference in their entirety for all purposes.

Additional PEG-lipids suitable for use in the invention include, withoutlimitation, mPEG2000-1,2-di-O-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG).The synthesis of PEG-C-DOMG is described in PCT Publication No. WO09/086558, the disclosure of which is herein incorporated by referencein its entirety for all purposes. Yet additional suitable PEG-lipidconjugates include, without limitation,1-[8′-(1,2-dimyristoyl-3-propanoxy)-carboxamido-3′,6′-dioxaoctanyl]carbamoyl-w-methyl-poly(ethyleneglycol) (2KPEG-DMG). The synthesis of 2KPEG-DMG is described in U.S.Pat. No. 7,404,969, the disclosure of which is herein incorporated byreference in its entirety for all purposes.

PEG is a linear, water-soluble polymer of ethylene PEG repeating unitswith two terminal hydroxyl groups. PEGs are classified by theirmolecular weights; for example, PEG 2000 has an average molecular weightof about 2,000 daltons, and PEG 5000 has an average molecular weight ofabout 5,000 daltons. PEGs are commercially available from Sigma ChemicalCo. and other companies and include, but are not limited to, thefollowing: monomethoxypolyethylene glycol (MePEG-OH),monomethoxypolyethylene glycol-succinate (MePEG-S),monomethoxypolyethylene glycol-succinimidyl succinate (MePEG-S—NHS),monomethoxypolyethylene glycol-amine (MePEG-NH₂),monomethoxypolyethylene glycol-tresylate (MePEG-TRES),monomethoxypolyethylene glycol-imidazolyl-carbonyl (MePEG-IM), as wellas such compounds containing a terminal hydroxyl group instead of aterminal methoxy group (e.g., HO-PEG-S, HO-PEG-S—NHS, HO-PEG-NH₂, etc.).Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing thePEG-lipid conjugates of the present invention. The disclosures of thesepatents are herein incorporated by reference in their entirety for allpurposes. In addition, monomethoxypolyethyleneglycol-acetic acid(MePEG-CH₂COOH) is particularly useful for preparing PEG-lipidconjugates including, e.g., PEG-DAA conjugates.

The PEG moiety of the PEG-lipid conjugates described herein may comprisean average molecular weight ranging from about 550 daltons to about10,000 daltons. In certain instances, the PEG moiety has an averagemolecular weight of from about 750 daltons to about 5,000 daltons (e.g.,from about 1,000 daltons to about 5,000 daltons, from about 1,500daltons to about 3,000 daltons, from about 750 daltons to about 3,000daltons, from about 750 daltons to about 2,000 daltons, etc.). Inpreferred embodiments, the PEG moiety has an average molecular weight ofabout 2,000 daltons or about 750 daltons.

In certain instances, the PEG can be optionally substituted by an alkyl,alkoxy, acyl, or aryl group. The PEG can be conjugated directly to thelipid or may be linked to the lipid via a linker moiety. Any linkermoiety suitable for coupling the PEG to a lipid can be used including,e.g., non-ester containing linker moieties and ester-containing linkermoieties. In a preferred embodiment, the linker moiety is a non-estercontaining linker moiety. As used herein, the term “non-ester containinglinker moiety” refers to a linker moiety that does not contain acarboxylic ester bond (—OC(O)—). Suitable non-ester containing linkermoieties include, but are not limited to, amido (—C(O)NH—), amino(—NR—), carbonyl (—C(O)—), carbamate (—NHC(O)O—), urea (—NHC(O)NH—),disulphide (—S—S—), ether (—O—), succinyl (—(O)CCH₂CH₂C(O)—),succinamidyl (—NHC(O)CH₂CH₂C(O)NH—), ether, disulphide, as well ascombinations thereof (such as a linker containing both a carbamatelinker moiety and an amido linker moiety). In a preferred embodiment, acarbamate linker is used to couple the PEG to the lipid.

In other embodiments, an ester containing linker moiety is used tocouple the PEG to the lipid. Suitable ester containing linker moietiesinclude, e.g., carbonate (—OC(O)O—), succinoyl, phosphate esters(—O—(O)POH—O—), sulfonate esters, and combinations thereof.

Phosphatidylethanolamines having a variety of acyl chain groups ofvarying chain lengths and degrees of saturation can be conjugated to PEGto form the lipid conjugate. Such phosphatidylethanolamines arecommercially available, or can be isolated or synthesized usingconventional techniques known to those of skilled in the art.Phosphatidyl-ethanolamines containing saturated or unsaturated fattyacids with carbon chain lengths in the range of C₁₀ to C₂₀ arepreferred. Phosphatidylethanolamines with mono- or diunsaturated fattyacids and mixtures of saturated and unsaturated fatty acids can also beused. Suitable phosphatidylethanolamines include, but are not limitedto, dimyristoyl-phosphatidylethanolamine (DMPE),dipalmitoyl-phosphatidylethanolamine (DPPE),dioleoylphosphatidylethanolamine (DOPE), anddistearoyl-phosphatidylethanolamine (DSPE).

The term “ATTA” or “polyamide” includes, without limitation, compoundsdescribed in U.S. Pat. Nos. 6,320,017 and 6,586,559, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes. These compounds include a compound having the formula:

wherein R is a member selected from the group consisting of hydrogen,alkyl and acyl; R¹ is a member selected from the group consisting ofhydrogen and alkyl; or optionally, R and R¹ and the nitrogen to whichthey are bound form an azido moiety; R² is a member of the groupselected from hydrogen, optionally substituted alkyl, optionallysubstituted aryl and a side chain of an amino acid; R³ is a memberselected from the group consisting of hydrogen, halogen, hydroxy,alkoxy, mercapto, hydrazino, amino and NR⁴R⁵, wherein R⁴ and R⁵ areindependently hydrogen or alkyl; n is 4 to 80; m is 2 to 6; p is 1 to 4;and q is 0 or 1. It will be apparent to those of skill in the art thatother polyamides can be used in the compounds of the present invention.

The term “diacylglycerol” or “DAG” includes a compound having 2 fattyacyl chains, R¹ and R², both of which have independently between 2 and30 carbons bonded to the 1- and 2-position of glycerol by esterlinkages. The acyl groups can be saturated or have varying degrees ofunsaturation. Suitable acyl groups include, but are not limited to,lauroyl (C₁₂), myristoyl (C₁₄), palmitoyl (C₁₆), stearoyl (C₁₈), andicosoyl (C₂₀). In preferred embodiments, R¹ and R² are the same, i.e.,R¹ and R² are both myristoyl (i.e., dimyristoyl), R¹ and R² are bothstearoyl (i.e., distearoyl), etc. Diacylglycerols have the followinggeneral formula:

The term “dialkyloxypropyl” or “DAA” includes a compound having 2 alkylchains, R¹ and R², both of which have independently between 2 and 30carbons. The alkyl groups can be saturated or have varying degrees ofunsaturation. Dialkyloxypropyls have the following general formula:

In a preferred embodiment, the PEG-lipid is a PEG-DAA conjugate havingthe following formula:

wherein R¹ and R² are independently selected and are long-chain alkylgroups having from about 10 to about 22 carbon atoms; PEG is apolyethyleneglycol; and L is a non-ester containing linker moiety or anester containing linker moiety as described above. The long-chain alkylgroups can be saturated or unsaturated. Suitable alkyl groups include,but are not limited to, decyl (C₁₀), lauryl (C₁₂), myristyl (C₁₄),palmityl (C₁₆), stearyl (C₁₈), and icosyl (C₂₀). In preferredembodiments, R¹ and R² are the same, i.e., R¹ and R² are both myristyl(i.e., dimyristyl), R¹ and R² are both stearyl (i.e., distearyl), etc.

In Formula V above, the PEG has an average molecular weight ranging fromabout 550 daltons to about 10,000 daltons. In certain instances, the PEGhas an average molecular weight of from about 750 daltons to about 5,000daltons (e.g., from about 1,000 daltons to about 5,000 daltons, fromabout 1,500 daltons to about 3,000 daltons, from about 750 daltons toabout 3,000 daltons, from about 750 daltons to about 2,000 daltons,etc.). In preferred embodiments, the PEG has an average molecular weightof about 2,000 daltons or about 750 daltons. The PEG can be optionallysubstituted with alkyl, alkoxy, acyl, or aryl groups. In certainembodiments, the terminal hydroxyl group is substituted with a methoxyor methyl group.

In a preferred embodiment, “L” is a non-ester containing linker moiety.Suitable non-ester containing linkers include, but are not limited to,an amido linker moiety, an amino linker moiety, a carbonyl linkermoiety, a carbamate linker moiety, a urea linker moiety, an ether linkermoiety, a disulphide linker moiety, a succinamidyl linker moiety, andcombinations thereof. In a preferred embodiment, the non-estercontaining linker moiety is a carbamate linker moiety (i.e., a PEG-C-DAAconjugate). In another preferred embodiment, the non-ester containinglinker moiety is an amido linker moiety (i.e., a PEG-A-DAA conjugate).In yet another preferred embodiment, the non-ester containing linkermoiety is a succinamidyl linker moiety (i.e., a PEG-S-DAA conjugate).

In particular embodiments, the PEG-lipid conjugate is selected from:

The PEG-DAA conjugates are synthesized using standard techniques andreagents known to those of skill in the art. It will be recognized thatthe PEG-DAA conjugates will contain various amide, amine, ether, thio,carbamate, and urea linkages. Those of skill in the art will recognizethat methods and reagents for forming these bonds are well known andreadily available. See, e.g., March, ADVANCED ORGANIC CHEMISTRY (Wiley1992); Larock, COMPREHENSIVE ORGANIC TRANSFORMATIONS (VCH 1989); andFurniss, VOGEL'S TEXTBOOK OF PRACTICAL ORGANIC CHEMISTRY, 5th ed.(Longman 1989). It will also be appreciated that any functional groupspresent may require protection and deprotection at different points inthe synthesis of the PEG-DAA conjugates. Those of skill in the art willrecognize that such techniques are well known. See, e.g., Green andWuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS (Wiley 1991).

Preferably, the PEG-DAA conjugate is a PEG-didecyloxypropyl (C₁₀)conjugate, a PEG-dilauryloxypropyl (C₁₂) conjugate, aPEG-dimyristyloxypropyl (C₁₄) conjugate, a PEG-dipalmityloxypropyl (C₁₆)conjugate, or a PEG-distearyloxypropyl (C₁₈) conjugate. In theseembodiments, the PEG preferably has an average molecular weight of about750 or about 2,000 daltons. In one particularly preferred embodiment,the PEG-lipid conjugate comprises PEG2000-C-DMA, wherein the “2000”denotes the average molecular weight of the PEG, the “C” denotes acarbamate linker moiety, and the “DMA” denotes dimyristyloxypropyl. Inanother particularly preferred embodiment, the PEG-lipid conjugatecomprises PEG750-C-DMA, wherein the “750” denotes the average molecularweight of the PEG, the “C” denotes a carbamate linker moiety, and the“DMA” denotes dimyristyloxypropyl. In particular embodiments, theterminal hydroxyl group of the PEG is substituted with a methyl group.Those of skill in the art will readily appreciate that otherdialkyloxypropyls can be used in the PEG-DAA conjugates of the presentinvention.

In addition to the foregoing, it will be readily apparent to those ofskill in the art that other hydrophilic polymers can be used in place ofPEG. Examples of suitable polymers that can be used in place of PEGinclude, but are not limited to, polyvinylpyrrolidone,polymethyloxazoline, polyethyloxazoline, polyhydroxypropylmethacrylamide, polymethacrylamide and polydimethylacrylamide,polylactic acid, polyglycolic acid, and derivatized celluloses such ashydroxymethylcellulose or hydroxyethylcellulose.

In addition to the foregoing components, the lipid particles (e.g.,SNALP) of the present invention can further comprise cationicpoly(ethylene glycol) (PEG) lipids or CPLs (see, e.g., Chen et al.,Bioconj. Chem., 11:433-437 (2000); U.S. Pat. No. 6,852,334; PCTPublication No. WO 00/62813, the disclosures of which are hereinincorporated by reference in their entirety for all purposes).

Suitable CPLs include compounds of Formula VI:

A-W—Y  (VI),

wherein A, W, and Y are as described below.

With reference to Formula VI, “A” is a lipid moiety such as anamphipathic lipid, a neutral lipid, or a hydrophobic lipid that acts asa lipid anchor. Suitable lipid examples include, but are not limited to,diacylglycerolyls, dialkylglycerolyls, N—N-dialkylaminos,1,2-diacyloxy-3-aminopropanes, and 1,2-dialkyl-3-aminopropanes.

“W” is a polymer or an oligomer such as a hydrophilic polymer oroligomer. Preferably, the hydrophilic polymer is a biocompatable polymerthat is nonimmunogenic or possesses low inherent immunogenicity.Alternatively, the hydrophilic polymer can be weakly antigenic if usedwith appropriate adjuvants. Suitable nonimmunogenic polymers include,but are not limited to, PEG, polyamides, polylactic acid, polyglycolicacid, polylactic acid/polyglycolic acid copolymers, and combinationsthereof. In a preferred embodiment, the polymer has a molecular weightof from about 250 to about 7,000 daltons.

“Y” is a polycationic moiety. The term polycationic moiety refers to acompound, derivative, or functional group having a positive charge,preferably at least 2 positive charges at a selected pH, preferablyphysiological pH. Suitable polycationic moieties include basic aminoacids and their derivatives such as arginine, asparagine, glutamine,lysine, and histidine; spermine; spermidine; cationic dendrimers;polyamines; polyamine sugars; and amino polysaccharides. Thepolycationic moieties can be linear, such as linear tetralysine,branched or dendrimeric in structure. Polycationic moieties have betweenabout 2 to about 15 positive charges, preferably between about 2 toabout 12 positive charges, and more preferably between about 2 to about8 positive charges at selected pH values. The selection of whichpolycationic moiety to employ may be determined by the type of particleapplication which is desired.

The charges on the polycationic moieties can be either distributedaround the entire particle moiety, or alternatively, they can be adiscrete concentration of charge density in one particular area of theparticle moiety e.g., a charge spike. If the charge density isdistributed on the particle, the charge density can be equallydistributed or unequally distributed. All variations of chargedistribution of the polycationic moiety are encompassed by the presentinvention.

The lipid “A” and the nonimmunogenic polymer “W” can be attached byvarious methods and preferably by covalent attachment. Methods known tothose of skill in the art can be used for the covalent attachment of “A”and “W.” Suitable linkages include, but are not limited to, amide,amine, carboxyl, carbonate, carbamate, ester, and hydrazone linkages. Itwill be apparent to those skilled in the art that “A” and “W” must havecomplementary functional groups to effectuate the linkage. The reactionof these two groups, one on the lipid and the other on the polymer, willprovide the desired linkage. For example, when the lipid is adiacylglycerol and the terminal hydroxyl is activated, for instance withNHS and DCC, to form an active ester, and is then reacted with a polymerwhich contains an amino group, such as with a polyamide (see, e.g., U.S.Pat. Nos. 6,320,017 and 6,586,559, the disclosures of which are hereinincorporated by reference in their entirety for all purposes), an amidebond will form between the two groups.

In certain instances, the polycationic moiety can have a ligandattached, such as a targeting ligand or a chelating moiety forcomplexing calcium. Preferably, after the ligand is attached, thecationic moiety maintains a positive charge. In certain instances, theligand that is attached has a positive charge. Suitable ligands include,but are not limited to, a compound or device with a reactive functionalgroup and include lipids, amphipathic lipids, carrier compounds,bioaffinity compounds, biomaterials, biopolymers, biomedical devices,analytically detectable compounds, therapeutically active compounds,enzymes, peptides, proteins, antibodies, immune stimulators,radiolabels, fluorogens, biotin, drugs, haptens, DNA, RNA,polysaccharides, liposomes, virosomes, micelles, immunoglobulins,functional groups, other targeting moieties, or toxins.

In some embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0.1 mol % to about 2 mol %, from about 0.5 mol % to about 2mol %, from about 1 mol % to about 2 mol %, from about 0.6 mol % toabout 1.9 mol %, from about 0.7 mol % to about 1.8 mol %, from about 0.8mol % to about 1.7 mol %, from about 0.9 mol % to about 1.6 mol %, fromabout 0.9 mol % to about 1.8 mol %, from about 1 mol % to about 1.8 mol%, from about 1 mol % to about 1.7 mol %, from about 1.2 mol % to about1.8 mol %, from about 1.2 mol % to about 1.7 mol %, from about 1.3 mol %to about 1.6 mol %, or from about 1.4 mol % to about 1.5 mol % (or anyfraction thereof or range therein) of the total lipid present in theparticle.

In other embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 0 mol % to about 20 mol %, from about 0.5 mol % to about 20mol %, from about 2 mol % to about 20 mol %, from about 1.5 mol % toabout 18 mol %, from about 2 mol % to about 15 mol %, from about 4 mol %to about 15 mol %, from about 2 mol % to about 12 mol %, from about 5mol % to about 12 mol %, or about 2 mol % (or any fraction thereof orrange therein) of the total lipid present in the particle.

In further embodiments, the lipid conjugate (e.g., PEG-lipid) comprisesfrom about 4 mol % to about 10 mol %, from about 5 mol % to about 10 mol%, from about 5 mol % to about 9 mol %, from about 5 mol % to about 8mol %, from about 6 mol % to about 9 mol %, from about 6 mol % to about8 mol %, or about 5 mol %, 6 mol %, 7 mol %, 8 mol %, 9 mol %, or 10 mol% (or any fraction thereof or range therein) of the total lipid presentin the particle.

Additional examples, percentages, and/or ranges of lipid conjugatessuitable for use in the lipid particles of the present invention aredescribed in, e.g., PCT Publication No. WO 09/127060, U.S. ProvisionalApplication No. 61/184,652, filed Jun. 5, 2009, U.S. ProvisionalApplication No. 61/295,134, filed Jan. 14, 2010, U.S. ProvisionalApplication No. 61/222,469, filed Jul. 1, 2009, U.S. ProvisionalApplication No. 61/294,828, filed Jan. 13, 2010, U.S. ProvisionalApplication No. 61/295,140, filed Jan. 14, 2010, and PCT Publication No.WO 2010/006282, the disclosures of which are herein incorporated byreference in their entirety for all purposes.

It should be understood that the percentage of lipid conjugate (e.g.,PEG-lipid) present in the lipid particles of the invention is a targetamount, and that the actual amount of lipid conjugate present in theformulation may vary, for example, by ±2 mol %. For example, in the 1:57lipid particle (e.g., SNALP) formulation, the target amount of lipidconjugate is 1.4 mol %, but the actual amount of lipid conjugate may be±0.5 mol %, ±0.4 mol %, ±0.3 mol %, ±0.2 mol %, ±0.1 mol %, or ±0.05 mol% of that target amount, with the balance of the formulation being madeup of other lipid components (adding up to 100 mol % of total lipidspresent in the particle). Similarly, in the 7:54 lipid particle (e.g.,SNALP) formulation, the target amount of lipid conjugate is 6.76 mol %,but the actual amount of lipid conjugate may be ±2 mol %, ±1.5 mol %, ±1mol %, ±0.75 mol %, ±0.5 mol %, ±0.25 mol %, or ±0.1 mol % of thattarget amount, with the balance of the formulation being made up ofother lipid components (adding up to 100 mol % of total lipids presentin the particle).

One of ordinary skill in the art will appreciate that the concentrationof the lipid conjugate can be varied depending on the lipid conjugateemployed and the rate at which the lipid particle is to becomefusogenic.

By controlling the composition and concentration of the lipid conjugate,one can control the rate at which the lipid conjugate exchanges out ofthe lipid particle and, in turn, the rate at which the lipid particlebecomes fusogenic. For instance, when a PEG-DAA conjugate is used as thelipid conjugate, the rate at which the lipid particle becomes fusogeniccan be varied, for example, by varying the concentration of the lipidconjugate, by varying the molecular weight of the PEG, or by varying thechain length and degree of saturation of the alkyl groups on the PEG-DAAconjugate. In addition, other variables including, for example, pH,temperature, ionic strength, etc. can be used to vary and/or control therate at which the lipid particle becomes fusogenic. Other methods whichcan be used to control the rate at which the lipid particle becomesfusogenic will become apparent to those of skill in the art upon readingthis disclosure. Also, by controlling the composition and concentrationof the lipid conjugate, one can control the lipid particle (e.g., SNALP)size.

VI. Preparation of Lipid Particles

The lipid particles of the present invention, e.g., SNALP, in which anactive agent or therapeutic agent such as an interfering RNA (e.g.,siRNA) is entrapped within the lipid portion of the particle and isprotected from degradation, can be formed by any method known in the artincluding, but not limited to, a continuous mixing method, a directdilution process, and an in-line dilution process.

In particular embodiments, the cationic lipids may comprise one or moreof the cationic lipids described herein or salts thereof, alone or incombination with other cationic lipids. In other embodiments, thenon-cationic lipids are egg sphingomyelin (ESM),distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC),dipalmitoyl-phosphatidylcholine (DPPC),monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine,14:0 PE (1,2-dimyristoyl-phosphatidylethanolamine (DMPE)), 16:0 PE(1,2-dipalmitoyl-phosphatidylethanolamine (DPPE)), 18:0 PE(1,2-distearoyl-phosphatidylethanolamine (DSPE)), 18:1 PE(1,2-dioleoyl-phosphatidylethanolamine (DOPE)), 18:1 trans PE(1,2-dielaidoyl-phosphatidylethanolamine (DEPE)), 18:0-18:1 PE(1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE)), 16:0-18:1 PE(1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE)), polyethyleneglycol-based polymers (e.g., PEG 2000, PEG 5000, PEG-modifieddiacylglycerols, or PEG-modified dialkyloxypropyls), cholesterol,derivatives thereof, or combinations thereof.

In a preferred embodiment, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via a direct dilutionprocess that includes forming a lipid vesicle (e.g., a non-lamellarlipid particle) solution and immediately and directly introducing thelipid vesicle solution into a collection vessel containing a controlledamount of dilution buffer. In preferred aspects, the collection vesselincludes one or more elements configured to stir the contents of thecollection vessel to facilitate dilution. In one aspect, the amount ofdilution buffer present in the collection vessel is substantially equalto the volume of lipid vesicle solution introduced thereto. As anon-limiting example, a lipid vesicle solution in 45% ethanol whenintroduced into the collection vessel containing an equal volume ofdilution buffer will advantageously yield smaller particles.

In a preferred embodiment, the present invention provides nucleicacid-lipid particles (e.g., SNALP) produced via an in-line dilutionprocess in which a third reservoir containing dilution buffer is fluidlycoupled to a second mixing region. In this embodiment, the lipid vesicle(e.g., non-lamellar lipid particle) solution formed in a first mixingregion is immediately and directly mixed with dilution buffer in thesecond mixing region. In preferred aspects, the second mixing regionincludes a T-connector arranged so that the lipid vesicle solution andthe dilution buffer flows meet as opposing 180° flows; however,connectors providing shallower angles can be used, e.g., from about 27°to about 180° (e.g., about 90°). A pump mechanism delivers acontrollable flow of buffer to the second mixing region. In one aspect,the flow rate of dilution buffer provided to the second mixing region iscontrolled to be substantially equal to the flow rate of lipid vesiclesolution introduced thereto from the first mixing region. Thisembodiment advantageously allows for more control of the flow ofdilution buffer mixing with the lipid vesicle solution in the secondmixing region, and therefore also the concentration of lipid vesiclesolution in buffer throughout the second mixing process. Such control ofthe dilution buffer flow rate advantageously allows for small particlesize formation at reduced concentrations.

These processes and the apparatuses for carrying out these directdilution and in-line dilution processes are described in detail in U.S.Patent Publication No. 20070042031, the disclosure of which is hereinincorporated by reference in its entirety for all purposes.

The nucleic acid-lipid particles formed using the direct dilution andin-line dilution processes typically have a size of from about 30 nm toabout 150 nm, from about 40 nm to about 150 nm, from about 50 nm toabout 150 nm, from about 60 nm to about 130 nm, from about 70 nm toabout 110 nm, from about 70 nm to about 100 nm, from about 80 nm toabout 100 nm, from about 90 nm to about 100 nm, from about 70 to about90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm,less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm,35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130nm, 135 nm, 140 nm, 145 nm, or 150 nm (or any fraction thereof or rangetherein). The particles thus formed do not aggregate and are optionallysized to achieve a uniform particle size.

In other embodiments, the present invention provides nucleic acid-lipidparticles (e.g., SNALP) produced via a continuous mixing method, e.g., aprocess that includes providing an aqueous solution comprising a nucleicacid (e.g., interfering RNA) in a first reservoir, providing an organiclipid solution in a second reservoir (wherein the lipids present in theorganic lipid solution are solubilized in an organic solvent, e.g., alower alkanol such as ethanol), and mixing the aqueous solution with theorganic lipid solution such that the organic lipid solution mixes withthe aqueous solution so as to substantially instantaneously produce alipid vesicle (e.g., liposome) encapsulating the nucleic acid within thelipid vesicle. This process and the apparatus for carrying out thisprocess are described in detail in U.S. Patent Publication No.20040142025, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

The action of continuously introducing lipid and buffer solutions into amixing environment, such as in a mixing chamber, causes a continuousdilution of the lipid solution with the buffer solution, therebyproducing a lipid vesicle substantially instantaneously upon mixing. Asused herein, the phrase “continuously diluting a lipid solution with abuffer solution” (and variations) generally means that the lipidsolution is diluted sufficiently rapidly in a hydration process withsufficient force to effectuate vesicle generation. By mixing the aqueoussolution comprising a nucleic acid with the organic lipid solution, theorganic lipid solution undergoes a continuous stepwise dilution in thepresence of the buffer solution (i.e., aqueous solution) to produce anucleic acid-lipid particle.

The nucleic acid-lipid particles formed using the continuous mixingmethod typically have a size of from about 30 nm to about 150 nm, fromabout 40 nm to about 150 nm, from about 50 nm to about 150 nm, fromabout 60 nm to about 130 nm, from about 70 nm to about 110 nm, fromabout 70 nm to about 100 nm, from about 80 nm to about 100 nm, fromabout 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80nm to about 90 nm, from about 70 nm to about 80 nm, less than about 120nm, 110 nm, 100 nm, 90 nm, or 80 nm, or about 30 nm, 35 nm, 40 nm, 45nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140nm, 145 nm, or 150 nm (or any fraction thereof or range therein). Theparticles thus formed do not aggregate and are optionally sized toachieve a uniform particle size.

If needed, the lipid particles of the invention (e.g., SNALP) can besized by any of the methods available for sizing liposomes. The sizingmay be conducted in order to achieve a desired size range and relativelynarrow distribution of particle sizes.

Several techniques are available for sizing the particles to a desiredsize. One sizing method, used for liposomes and equally applicable tothe present particles, is described in U.S. Pat. No. 4,737,323, thedisclosure of which is herein incorporated by reference in its entiretyfor all purposes. Sonicating a particle suspension either by bath orprobe sonication produces a progressive size reduction down to particlesof less than about 50 nm in size. Homogenization is another method whichrelies on shearing energy to fragment larger particles into smallerones. In a typical homogenization procedure, particles are recirculatedthrough a standard emulsion homogenizer until selected particle sizes,typically between about 60 and about 80 nm, are observed. In bothmethods, the particle size distribution can be monitored by conventionallaser-beam particle size discrimination, or QELS.

Extrusion of the particles through a small-pore polycarbonate membraneor an asymmetric ceramic membrane is also an effective method forreducing particle sizes to a relatively well-defined size distribution.Typically, the suspension is cycled through the membrane one or moretimes until the desired particle size distribution is achieved. Theparticles may be extruded through successively smaller-pore membranes,to achieve a gradual reduction in size.

In some embodiments, the nucleic acids present in the particles areprecondensed as described in, e.g., U.S. patent application Ser. No.09/744,103, the disclosure of which is herein incorporated by referencein its entirety for all purposes.

In other embodiments, the methods may further comprise adding non-lipidpolycations which are useful to effect the lipofection of cells usingthe present compositions. Examples of suitable non-lipid polycationsinclude, hexadimethrine bromide (sold under the brand name POLYBRENE®,from Aldrich Chemical Co., Milwaukee, Wis., USA) or other salts ofhexadimethrine. Other suitable polycations include, for example, saltsof poly-L-ornithine, poly-L-arginine, poly-L-lysine, poly-D-lysine,polyallylamine, and polyethyleneimine. Addition of these salts ispreferably after the particles have been formed.

In some embodiments, the nucleic acid to lipid ratios (mass/mass ratios)in a formed nucleic acid-lipid particle (e.g., SNALP) will range fromabout 0.01 to about 0.2, from about 0.05 to about 0.2, from about 0.02to about 0.1, from about 0.03 to about 0.1, or from about 0.01 to about0.08. The ratio of the starting materials (input) also falls within thisrange. In other embodiments, the particle preparation uses about 400 μgnucleic acid per 10 mg total lipid or a nucleic acid to lipid mass ratioof about 0.01 to about 0.08 and, more preferably, about 0.04, whichcorresponds to 1.25 mg of total lipid per 50 μg of nucleic acid. Inother preferred embodiments, the particle has a nucleic acid:lipid massratio of about 0.08.

In other embodiments, the lipid to nucleic acid ratios (mass/massratios) in a formed nucleic acid-lipid particle (e.g., SNALP) will rangefrom about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100(100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) toabout 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4(4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), fromabout 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1),from about 3 (3:1) to about 25 (25:1), from about 4 (4:1) to about 25(25:1), from about 5 (5:1) to about 25 (25:1), from about 5 (5:1) toabout 20 (20:1), from about 5 (5:1) to about 15 (15:1), from about 5(5:1) to about 10 (10:1), or about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9(9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), 15 (15:1),16 (16:1), 17 (17:1), 18 (18:1), 19 (19:1), 20 (20:1), 21 (21:1), 22(22:1), 23 (23:1), 24 (24:1), or 25 (25:1), or any fraction thereof orrange therein. The ratio of the starting materials (input) also fallswithin this range.

As previously discussed, the conjugated lipid may further include a CPL.A variety of general methods for making SNALP-CPLs (CPL-containingSNALP) are discussed herein. Two general techniques include the“post-insertion” technique, that is, insertion of a CPL into, forexample, a pre-formed SNALP, and the “standard” technique, wherein theCPL is included in the lipid mixture during, for example, the SNALPformation steps. The post-insertion technique results in SNALP havingCPLs mainly in the external face of the SNALP bilayer membrane, whereasstandard techniques provide SNALP having CPLs on both internal andexternal faces. The method is especially useful for vesicles made fromphospholipids (which can contain cholesterol) and also for vesiclescontaining PEG-lipids (such as PEG-DAAs and PEG-DAGs). Methods of makingSNALP-CPLs are taught, for example, in U.S. Pat. Nos. 5,705,385;6,586,410; 5,981,501; 6,534,484; and 6,852,334; U.S. Patent PublicationNo. 20020072121; and PCT Publication No. WO 00/62813, the disclosures ofwhich are herein incorporated by reference in their entirety for allpurposes.

VII. Kits

The present invention also provides lipid particles (e.g., SNALP) in kitform. In some embodiments, the kit comprises a container which iscompartmentalized for holding the various elements of the lipidparticles (e.g., the active agents or therapeutic agents such as nucleicacids and the individual lipid components of the particles). Preferably,the kit comprises a container (e.g., a vial or ampoule) which holds thelipid particles of the invention (e.g., SNALP), wherein the particlesare produced by one of the processes set forth herein. In certainembodiments, the kit may further comprise an endosomal membranedestabilizer (e.g., calcium ions). The kit typically contains theparticle compositions of the invention, either as a suspension in apharmaceutically acceptable carrier or in dehydrated form, withinstructions for their rehydration (if lyophilized) and administration.

The lipid particles of the present invention can be tailored topreferentially target particular tissues, organs, or tumors of interest.In some instances, the 1:57 lipid particle (e.g., SNALP) formulation canbe used to preferentially target the liver (e.g., normal liver tissue).In other instances, the 7:54 lipid particle (e.g., SNALP) formulationcan be used to preferentially target solid tumors such as liver tumorsand tumors outside of the liver. In preferred embodiments, the kits ofthe invention comprise these liver-directed and/or tumor-directed lipidparticles, wherein the particles are present in a container as asuspension or in dehydrated form.

In certain other instances, it may be desirable to have a targetingmoiety attached to the surface of the lipid particle to further enhancethe targeting of the particle. Methods of attaching targeting moieties(e.g., antibodies, proteins, etc.) to lipids (such as those used in thepresent particles) are known to those of skill in the art.

VIII. Administration of Lipid Particles

Once formed, the lipid particles of the invention (e.g., SNALP) areuseful for the introduction of active agents or therapeutic agents(e.g., nucleic acids such as interfering RNA) into cells. Accordingly,the present invention also provides methods for introducing an activeagent or therapeutic agent such as a nucleic acid (e.g., interferingRNA) into a cell. In some instances, the cell is a liver cell such as,e.g., a hepatocyte present in liver tissue. In other instances, the cellis a tumor cell such as, e.g., a tumor cell present in a solid tumor.The methods are carried out in vitro or in vivo by first forming theparticles as described above and then contacting the particles with thecells for a period of time sufficient for delivery of the active agentor therapeutic agent to the cells to occur.

The lipid particles of the invention (e.g., SNALP) can be adsorbed toalmost any cell type with which they are mixed or contacted. Onceadsorbed, the particles can either be endocytosed by a portion of thecells, exchange lipids with cell membranes, or fuse with the cells.Transfer or incorporation of the active agent or therapeutic agent(e.g., nucleic acid) portion of the particle can take place via any oneof these pathways. In particular, when fusion takes place, the particlemembrane is integrated into the cell membrane and the contents of theparticle combine with the intracellular fluid.

The lipid particles of the invention (e.g., SNALP) can be administeredeither alone or in a mixture with a pharmaceutically acceptable carrier(e.g., physiological saline or phosphate buffer) selected in accordancewith the route of administration and standard pharmaceutical practice.Generally, normal buffered saline (e.g., 135-150 mM NaCl) will beemployed as the pharmaceutically acceptable carrier. Other suitablecarriers include, e.g., water, buffered water, 0.4% saline, 0.3%glycine, and the like, including glycoproteins for enhanced stability,such as albumin, lipoprotein, globulin, etc. Additional suitablecarriers are described in, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES,Mack Publishing Company, Philadelphia, Pa., 17th ed. (1985). As usedherein, “carrier” includes any and all solvents, dispersion media,vehicles, coatings, diluents, antibacterial and antifungal agents,isotonic and absorption delaying agents, buffers, carrier solutions,suspensions, colloids, and the like. The phrase “pharmaceuticallyacceptable” refers to molecular entities and compositions that do notproduce an allergic or similar untoward reaction when administered to ahuman.

The pharmaceutically acceptable carrier is generally added followinglipid particle formation. Thus, after the lipid particle (e.g., SNALP)is formed, the particle can be diluted into pharmaceutically acceptablecarriers such as normal buffered saline.

The concentration of particles in the pharmaceutical formulations canvary widely, i.e., from less than about 0.05%, usually at or at leastabout 2 to 5%, to as much as about 10 to 90% by weight, and will beselected primarily by fluid volumes, viscosities, etc., in accordancewith the particular mode of administration selected. For example, theconcentration may be increased to lower the fluid load associated withtreatment. This may be particularly desirable in patients havingatherosclerosis-associated congestive heart failure or severehypertension. Alternatively, particles composed of irritating lipids maybe diluted to low concentrations to lessen inflammation at the site ofadministration.

The pharmaceutical compositions of the present invention may besterilized by conventional, well-known sterilization techniques. Aqueoussolutions can be packaged for use or filtered under aseptic conditionsand lyophilized, the lyophilized preparation being combined with asterile aqueous solution prior to administration. The compositions cancontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, andcalcium chloride. Additionally, the particle suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as alphatocopherol, and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

In some embodiments, the lipid particles of the invention (e.g., SNALP)are particularly useful in methods for the therapeutic delivery of oneor more nucleic acids comprising an interfering RNA sequence (e.g.,siRNA). In particular, it is an object of this invention to provide invitro and in vivo methods for treatment of a disease or disorder in amammal (e.g., a rodent such as a mouse or a primate such as a human,chimpanzee, or monkey) by downregulating or silencing the transcriptionand/or translation of one or more target nucleic acid sequences or genesof interest. As a non-limiting example, the methods of the invention areuseful for in vivo delivery of interfering RNA (e.g., siRNA) to theliver and/or tumor of a mammalian subject. In certain embodiments, thedisease or disorder is associated with expression and/or overexpressionof a gene and expression or overexpression of the gene is reduced by theinterfering RNA (e.g., siRNA). In certain other embodiments, atherapeutically effective amount of the lipid particle may beadministered to the mammal. In some instances, an interfering RNA (e.g.,siRNA) is formulated into a SNALP, and the particles are administered topatients requiring such treatment. In other instances, cells are removedfrom a patient, the interfering RNA is delivered in vitro (e.g., using aSNALP described herein), and the cells are reinjected into the patient.

A. In Vivo Administration

Systemic delivery for in vivo therapy, e.g., delivery of a therapeuticnucleic acid to a distal target cell via body systems such as thecirculation, has been achieved using nucleic acid-lipid particles suchas those described in PCT Publication Nos. WO 05/007196, WO 05/121348,WO 05/120152, and WO 04/002453, the disclosures of which are hereinincorporated by reference in their entirety for all purposes. Thepresent invention also provides fully encapsulated lipid particles thatprotect the nucleic acid from nuclease degradation in serum, arenon-immunogenic, are small in size, and are suitable for repeat dosing.

For in vivo administration, administration can be in any manner known inthe art, e.g., by injection, oral administration, inhalation (e.g.,intransal or intratracheal), transdermal application, or rectaladministration. Administration can be accomplished via single or divideddoses. The pharmaceutical compositions can be administered parenterally,i.e., intraarticularly, intravenously, intraperitoneally,subcutaneously, or intramuscularly. In some embodiments, thepharmaceutical compositions are administered intravenously orintraperitoneally by a bolus injection (see, e.g., U.S. Pat. No.5,286,634). Intracellular nucleic acid delivery has also been discussedin Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino etal., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. DrugCarrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993).Still other methods of administering lipid-based therapeutics aredescribed in, for example, U.S. Pat. Nos. 3,993,754; 4,145,410;4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles canbe administered by direct injection at the site of disease or byinjection at a site distal from the site of disease (see, e.g., Culver,HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp.70-71(1994)). The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

In embodiments where the lipid particles of the present invention (e.g.,SNALP) are administered intravenously, at least about 5%, 10%, 15%, 20%,or 25% of the total injected dose of the particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In other embodiments,more than about 20%, 30%, 40% and as much as about 60%, 70% or 80% ofthe total injected dose of the lipid particles is present in plasmaabout 8, 12, 24, 36, or 48 hours after injection. In certain instances,more than about 10% of a plurality of the particles is present in theplasma of a mammal about 1 hour after administration. In certain otherinstances, the presence of the lipid particles is detectable at leastabout 1 hour after administration of the particle. In certainembodiments, the presence of a therapeutic agent such as a nucleic acidis detectable in cells of the lung, liver, tumor, or at a site ofinflammation at about 8, 12, 24, 36, 48, 60, 72 or 96 hours afteradministration. In other embodiments, downregulation of expression of atarget sequence by an interfering RNA (e.g., siRNA) is detectable atabout 8, 12, 24, 36, 48, 60, 72 or 96 hours after administration. In yetother embodiments, downregulation of expression of a target sequence byan interfering RNA (e.g., siRNA) occurs preferentially in liver cells(e.g., hepatocytes), tumor cells, or in cells at a site of inflammation.In further embodiments, the presence or effect of an interfering RNA(e.g., siRNA) in cells at a site proximal or distal to the site ofadministration or in cells of the lung, liver, or a tumor is detectableat about 12, 24, 48, 72, or 96 hours, or at about 6, 8, 10, 12, 14, 16,18, 19, 20, 22, 24, 26, or 28 days after administration. In additionalembodiments, the lipid particles (e.g., SNALP) of the invention areadministered parenterally or intraperitoneally.

The compositions of the present invention, either alone or incombination with other suitable components, can be made into aerosolformulations (i.e., they can be “nebulized”) to be administered viainhalation (e.g., intranasally or intratracheally) (see, Brigham et al.,Am. J. Sci., 298:278 (1989)). Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering nucleic acid compositions directly tothe lungs via nasal aerosol sprays have been described, e.g., in U.S.Pat. Nos. 5,756,353 and 5,804,212. Likewise, the delivery of drugs usingintranasal microparticle resins and lysophosphatidyl-glycerol compounds(U.S. Pat. No. 5,725,871) are also well-known in the pharmaceuticalarts. Similarly, transmucosal drug delivery in the form of apolytetrafluoroetheylene support matrix is described in U.S. Pat. No.5,780,045. The disclosures of the above-described patents are hereinincorporated by reference in their entirety for all purposes.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.In the practice of this invention, compositions are preferablyadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically, or intrathecally.

Generally, when administered intravenously, the lipid particleformulations are formulated with a suitable pharmaceutical carrier. Manypharmaceutically acceptable carriers may be employed in the compositionsand methods of the present invention. Suitable formulations for use inthe present invention are found, for example, in REMINGTON'SPHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, Pa.,17th ed. (1985). A variety of aqueous carriers may be used, for example,water, buffered water, 0.4% saline, 0.3% glycine, and the like, and mayinclude glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. Generally, normal buffered saline (135-150mM NaCl) will be employed as the pharmaceutically acceptable carrier,but other suitable carriers will suffice. These compositions can besterilized by conventional liposomal sterilization techniques, such asfiltration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as pH adjusting and buffering agents, tonicityadjusting agents, wetting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, sorbitan monolaurate, triethanolamine oleate, etc. Thesecompositions can be sterilized using the techniques referred to aboveor, alternatively, they can be produced under sterile conditions. Theresulting aqueous solutions may be packaged for use or filtered underaseptic conditions and lyophilized, the lyophilized preparation beingcombined with a sterile aqueous solution prior to administration.

In certain applications, the lipid particles disclosed herein may bedelivered via oral administration to the individual. The particles maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, pills, lozenges, elixirs,mouthwash, suspensions, oral sprays, syrups, wafers, and the like (see,e.g., U.S. Pat. Nos. 5,641,515, 5,580,579, and 5,792,451, thedisclosures of which are herein incorporated by reference in theirentirety for all purposes). These oral dosage forms may also contain thefollowing: binders, gelatin; excipients, lubricants, and/or flavoringagents. When the unit dosage form is a capsule, it may contain, inaddition to the materials described above, a liquid carrier. Variousother materials may be present as coatings or to otherwise modify thephysical form of the dosage unit. Of course, any material used inpreparing any unit dosage form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed.

Typically, these oral formulations may contain at least about 0.1% ofthe lipid particles or more, although the percentage of the particlesmay, of course, be varied and may conveniently be between about 1% or 2%and about 60% or 70% or more of the weight or volume of the totalformulation. Naturally, the amount of particles in each therapeuticallyuseful composition may be prepared is such a way that a suitable dosagewill be obtained in any given unit dose of the compound. Factors such assolubility, bioavailability, biological half-life, route ofadministration, product shelf life, as well as other pharmacologicalconsiderations will be contemplated by one skilled in the art ofpreparing such pharmaceutical formulations, and as such, a variety ofdosages and treatment regimens may be desirable.

Formulations suitable for oral administration can consist of: (a) liquidsolutions, such as an effective amount of a packaged therapeutic agentsuch as nucleic acid (e.g., interfering RNA) suspended in diluents suchas water, saline, or PEG 400; (b) capsules, sachets, or tablets, eachcontaining a predetermined amount of a therapeutic agent such as nucleicacid (e.g., interfering RNA), as liquids, solids, granules, or gelatin;(c) suspensions in an appropriate liquid; and (d) suitable emulsions.Tablet forms can include one or more of lactose, sucrose, mannitol,sorbitol, calcium phosphates, corn starch, potato starch,microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc,magnesium stearate, stearic acid, and other excipients, colorants,fillers, binders, diluents, buffering agents, moistening agents,preservatives, flavoring agents, dyes, disintegrating agents, andpharmaceutically compatible carriers. Lozenge forms can comprise atherapeutic agent such as nucleic acid (e.g., interfering RNA) in aflavor, e.g., sucrose, as well as pastilles comprising the therapeuticagent in an inert base, such as gelatin and glycerin or sucrose andacacia emulsions, gels, and the like containing, in addition to thetherapeutic agent, carriers known in the art.

In another example of their use, lipid particles can be incorporatedinto a broad range of topical dosage forms. For instance, a suspensioncontaining nucleic acid-lipid particles such as SNALP can be formulatedand administered as gels, oils, emulsions, topical creams, pastes,ointments, lotions, foams, mousses, and the like.

When preparing pharmaceutical preparations of the lipid particles of theinvention, it is preferable to use quantities of the particles whichhave been purified to reduce or eliminate empty particles or particleswith therapeutic agents such as nucleic acid associated with theexternal surface.

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as primates(e.g., humans and chimpanzees as well as other nonhuman primates),canines, felines, equines, bovines, ovines, caprines, rodents (e.g.,rats and mice), lagomorphs, and swine.

The amount of particles administered will depend upon the ratio oftherapeutic agent (e.g., nucleic acid) to lipid, the particulartherapeutic agent (e.g., nucleic acid) used, the disease or disorderbeing treated, the age, weight, and condition of the patient, and thejudgment of the clinician, but will generally be between about 0.01 andabout 50 mg per kilogram of body weight, preferably between about 0.1and about 5 mg/kg of body weight, or about 10⁸-10¹⁰ particles peradministration (e.g., injection).

B. In Vitro Administration

For in vitro applications, the delivery of therapeutic agents such asnucleic acids (e.g., interfering RNA) can be to any cell grown inculture, whether of plant or animal origin, vertebrate or invertebrate,and of any tissue or type. In preferred embodiments, the cells areanimal cells, more preferably mammalian cells, and most preferably humancells (e.g., tumor cells or hepatocytes).

Contact between the cells and the lipid particles, when carried out invitro, takes place in a biologically compatible medium. Theconcentration of particles varies widely depending on the particularapplication, but is generally between about 1 μmol and about 10 mmol.Treatment of the cells with the lipid particles is generally carried outat physiological temperatures (about 37° C.) for periods of time of fromabout 1 to 48 hours, preferably of from about 2 to 4 hours.

In one group of preferred embodiments, a lipid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/ml, more preferably about 2×10⁴ cells/ml.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 0.2 μg/ml, more preferably about 0.1 μg/ml.

To the extent that tissue culture of cells may be required, it iswell-known in the art. For example, Freshney, Culture of Animal Cells, aManual of Basic Technique, 3rd Ed., Wiley-Liss, New York (1994), Kuchleret al., Biochemical Methods in Cell Culture and Virology, Dowden,Hutchinson and Ross, Inc. (1977), and the references cited thereinprovide a general guide to the culture of cells. Cultured cell systemsoften will be in the form of monolayers of cells, although cellsuspensions are also used.

Using an Endosomal Release Parameter (ERP) assay, the deliveryefficiency of the SNALP or other lipid particle of the invention can beoptimized. An ERP assay is described in detail in U.S. PatentPublication No. 20030077829, the disclosure of which is hereinincorporated by reference in its entirety for all purposes. Moreparticularly, the purpose of an ERP assay is to distinguish the effectof various cationic lipids and helper lipid components of SNALP or otherlipid particle based on their relative effect on binding/uptake orfusion with/destabilization of the endosomal membrane. This assay allowsone to determine quantitatively how each component of the SNALP or otherlipid particle affects delivery efficiency, thereby optimizing the SNALPor other lipid particle. Usually, an ERP assay measures expression of areporter protein (e.g., luciferase, (3-galactosidase, green fluorescentprotein (GFP), etc.), and in some instances, a SNALP formulationoptimized for an expression plasmid will also be appropriate forencapsulating an interfering RNA. In other instances, an ERP assay canbe adapted to measure downregulation of transcription or translation ofa target sequence in the presence or absence of an interfering RNA(e.g., siRNA). By comparing the ERPs for each of the various SNALP orother lipid particles, one can readily determine the optimized system,e.g., the SNALP or other lipid particle that has the greatest uptake inthe cell.

C. Cells for Delivery of Lipid Particles

The compositions and methods of the present invention are used to treata wide variety of cell types, in vivo and in vitro. Suitable cellsinclude, but are not limited to, hepatocytes, reticuloendothelial cells(e.g., monocytes, macrophages, etc.), fibroblast cells, endothelialcells, platelet cells, other cell types infected and/or susceptible ofbeing infected with viruses, hematopoietic precursor (stem) cells,keratinocytes, skeletal and smooth muscle cells, osteoblasts, neurons,quiescent lymphocytes, terminally differentiated cells, slow ornoncycling primary cells, parenchymal cells, lymphoid cells, epithelialcells, bone cells, and the like.

In particular embodiments, an active agent or therapeutic agent such asa nucleic acid (e.g., an interfering RNA) is delivered to cancer cells(e.g., cells of a solid tumor) including, but not limited to, livercancer cells, lung cancer cells, colon cancer cells, rectal cancercells, anal cancer cells, bile duct cancer cells, small intestine cancercells, stomach (gastric) cancer cells, esophageal cancer cells,gallbladder cancer cells, pancreatic cancer cells, appendix cancercells, breast cancer cells, ovarian cancer cells, cervical cancer cells,prostate cancer cells, renal cancer cells, cancer cells of the centralnervous system, glioblastoma tumor cells, skin cancer cells, lymphomacells, choriocarcinoma tumor cells, head and neck cancer cells,osteogenic sarcoma tumor cells, and blood cancer cells.

In vivo delivery of lipid particles such as SNALP encapsulating anucleic acid (e.g., an interfering RNA) is suited for targeting cells ofany cell type. The methods and compositions can be employed with cellsof a wide variety of vertebrates, including mammals, such as, e.g.canines, felines, equines, bovines, ovines, caprines, rodents (e.g.,mice, rats, and guinea pigs), lagomorphs, swine, and primates (e.g.monkeys, chimpanzees, and humans).

D. Detection of Lipid Particles

In some embodiments, the lipid particles of the present invention (e.g.,SNALP) are detectable in the subject at about 1, 2, 3, 4, 5, 6, 7, 8 ormore hours. In other embodiments, the lipid particles of the presentinvention (e.g., SNALP) are detectable in the subject at about 8, 12,24, 48, 60, 72, or 96 hours, or about 6, 8, 10, 12, 14, 16, 18, 19, 22,24, 25, or 28 days after administration of the particles. The presenceof the particles can be detected in the cells, tissues, or otherbiological samples from the subject. The particles may be detected,e.g., by direct detection of the particles, detection of a therapeuticnucleic acid such as an interfering RNA (e.g., siRNA) sequence,detection of the target sequence of interest (i.e., by detectingexpression or reduced expression of the sequence of interest), or acombination thereof.

1. Detection of Particles

Lipid particles of the invention such as SNALP can be detected using anymethod known in the art. For example, a label can be coupled directly orindirectly to a component of the lipid particle using methods well-knownin the art. A wide variety of labels can be used, with the choice oflabel depending on sensitivity required, ease of conjugation with thelipid particle component, stability requirements, and availableinstrumentation and disposal provisions. Suitable labels include, butare not limited to, spectral labels such as fluorescent dyes (e.g.,fluorescein and derivatives, such as fluorescein isothiocyanate (FITC)and Oregon Green™; rhodamine and derivatives such Texas red,tetrarhodimine isothiocynate (TRITC), etc., digoxigenin, biotin,phycoerythrin, AMCA, CyDyes™, and the like; radiolabels such as ³H,¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.; enzymes such as horse radish peroxidase,alkaline phosphatase, etc.; spectral colorimetric labels such ascolloidal gold or colored glass or plastic beads such as polystyrene,polypropylene, latex, etc. The label can be detected using any meansknown in the art.

2. Detection of Nucleic Acids

Nucleic acids (e.g., interfering RNA) are detected and quantified hereinby any of a number of means well-known to those of skill in the art. Thedetection of nucleic acids may proceed by well-known methods such asSouthern analysis, Northern analysis, gel electrophoresis, PCR,radiolabeling, scintillation counting, and affinity chromatography.Additional analytic biochemical methods such as spectrophotometry,radiography, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), and hyperdiffusion chromatography may also be employed.

The selection of a nucleic acid hybridization format is not critical. Avariety of nucleic acid hybridization formats are known to those skilledin the art. For example, common formats include sandwich assays andcompetition or displacement assays. Hybridization techniques aregenerally described in, e.g., “Nucleic Acid Hybridization, A PracticalApproach,” Eds. Hames and Higgins, IRL Press (1985).

The sensitivity of the hybridization assays may be enhanced through theuse of a nucleic acid amplification system which multiplies the targetnucleic acid being detected. In vitro amplification techniques suitablefor amplifying sequences for use as molecular probes or for generatingnucleic acid fragments for subsequent subcloning are known. Examples oftechniques sufficient to direct persons of skill through such in vitroamplification methods, including the polymerase chain reaction (PCR),the ligase chain reaction (LCR), Qβ-replicase amplification, and otherRNA polymerase mediated techniques (e.g., NASBA™) are found in Sambrooket al., In Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press (2000); and Ausubel et al., SHORT PROTOCOLS INMOLECULAR BIOLOGY, eds., Current Protocols, Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (2002); as well as U.S.Pat. No. 4,683,202; PCR Protocols, A Guide to Methods and Applications(Innis et al. eds.) Academic Press Inc. San Diego, Calif. (1990);Arnheim & Levinson (Oct. 1, 1990), C&EN 36; The Journal Of NIH Research,3:81 (1991); Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989);Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874 (1990); Lomell etal., J. Clin. Chem., 35:1826 (1989); Landegren et al., Science, 241:1077(1988); Van Brunt, Biotechnology, 8:291 (1990); Wu and Wallace, Gene,4:560 (1989); Barringer et al., Gene, 89:117 (1990); and Sooknanan andMalek, Biotechnology, 13:563 (1995). Improved methods of cloning invitro amplified nucleic acids are described in U.S. Pat. No. 5,426,039.Other methods described in the art are the nucleic acid sequence basedamplification (NASBA™, Cangene, Mississauga, Ontario) and Qβ-replicasesystems. These systems can be used to directly identify mutants wherethe PCR or LCR primers are designed to be extended or ligated only whena select sequence is present. Alternatively, the select sequences can begenerally amplified using, for example, nonspecific PCR primers and theamplified target region later probed for a specific sequence indicativeof a mutation. The disclosures of the above-described references areherein incorporated by reference in their entirety for all purposes.

Nucleic acids for use as probes, e.g., in in vitro amplificationmethods, for use as gene probes, or as inhibitor components aretypically synthesized chemically according to the solid phasephosphoramidite triester method described by Beaucage et al.,Tetrahedron Letts., 22:1859 1862 (1981), e.g., using an automatedsynthesizer, as described in Needham VanDevanter et al., Nucleic AcidsRes., 12:6159 (1984). Purification of polynucleotides, where necessary,is typically performed by either native acrylamide gel electrophoresisor by anion exchange HPLC as described in Pearson et al., J. Chrom.,255:137 149 (1983). The sequence of the synthetic polynucleotides can beverified using the chemical degradation method of Maxam and Gilbert(1980) in Grossman and Moldave (eds.) Academic Press, New York, Methodsin Enzymology, 65:499.

An alternative means for determining the level of transcription is insitu hybridization. In situ hybridization assays are well-known and aregenerally described in Angerer et al., Methods Enzymol., 152:649 (1987).In an in situ hybridization assay, cells are fixed to a solid support,typically a glass slide. If DNA is to be probed, the cells are denaturedwith heat or alkali. The cells are then contacted with a hybridizationsolution at a moderate temperature to permit annealing of specificprobes that are labeled. The probes are preferably labeled withradioisotopes or fluorescent reporters.

IX. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

Example 1 Characterization of SNALP Structure

This example demonstrates that by controlling the lipid composition ofthe SNALP formulation as well as the particle formation process, novelnon-lamellar lipid nanoparticles (e.g., SNALP) can be produced that haveenhanced activity. In this example, SNALP formulations of varyingcompositions were prepared using either a Stepwise Dilution Method or aDirect Dilution Method to study the effects the manufacturing processand/or the lipid composition had on particle size, encapsulationefficiency and morphology.

The Stepwise Dilution Method (“SDM”), which is also referred to hereinas the Lipomixer I process, as well as the apparatuses for carrying outthe SDM are described in detail in U.S. Patent Publication No.20040142025 and in Jeffs, et al., “A Scalable, Extrusion-Free Method forEfficient Liposomal Encapsulation of Plasmid DNA,” PharmaceuticalResearch, 2005, Vol. 22, No. 3 pp 362-372, the disclosures of both ofwhich are herein incorporated by reference in their entirety for allpurposes. As illustrated in FIG. 1A, in the Stepwise Dilution method, anucleic acid solution (e.g., ApoB-8, 100 mM EDTA, nuclease-free water)is blended with an equal volume of warmed lipid stock solution in 90%ethanol at 75 mL/min using a Watson-Marlow peristaltic pump. The blendedsolution, i.e., Stabilized NALP in 45% ethanol, is further diluted withan equal volume of warmed citrate/NaCl buffer at 75 mL/min, resulting inStabilized NALP in 22.5% ethanol. The ethanol is removed from thestabilized NALP formulation by TFU hoop cartridges.

The Direct Dilution Method (“DDM”), which is also referred to herein asthe Lipomixer II process, as well as the apparatuses for carrying outthe DDM are described in detail in U.S. Patent Publication No.20070042031, the disclosure of which is herein incorporated by referencein its entirety for all purposes. As illustrated in FIG. 1B, in theDirect Dilution method, equal volumes of nucleic acid solution (e.g.,ApoB-8, 100 mM EDTA, nuclease-free water) and lipid stock solution wereblended at 200 mL/min using an automated syringe press (i.e.,“Lipobot”), and diluted directly into PBS. The ethanol is removed fromthe stabilized NALP formulation by TFU hoop cartridge.

Table 1 sets forth a comparison of the process parameters for theStepwise Dilution and Direct Dilution methods.

TABLE 1 Process Parameters for the Stepwise Dilution and Direct DilutionMethods SDM DDM Batch Size 5-10 mg 7-10 mg Lipid Stocks 90% ethanol 100%ethanol Equipment Peristaltic Pump Automated Syringe Press “Lipobot”Blending Rate 75 mL/min 200 mL/min T-connector 1.6 mm ID 200 mL/minDilution Buffer 20 nM citrate/ PBS, pH 7.4 300 mM NaCl, pH

All SNALP formulations used in this study were prepared with an siRNAtargeting apolipoprotein B (ApoB) as the nucleic acid component. ApoB isthe main apolipoprotein of chylomicrons and low density lipoproteins(LDL). Mutations in ApoB are associated with hypercholesterolemia. ApoBoccurs in the plasma in 2 main forms, ApoB48 and ApoB100, which aresynthesized in the intestine and liver, respectively, due to anorgan-specific stop codon. The ApoB siRNA used in this study is providedin Table 2, and is referred to herein as “siApoB-8.” The modificationsinvolved introducing 2′OMe-uridine or 2′OMe-guanosine at selectedpositions in the sense and antisense strands of the ApoB siRNA sequence,in which the siRNA duplex contained less than about 20% 2′OMe-modifiednucleotides.

TABLE 2siRNA duplex comprising sense and antisense ApoB RNA polynucleotides.% 2′OMe- % Modified Position Modification ApoB siRNA sequence Modifiedin DS Region 10048 U2/2 G1/2 5′-AGU G UCA U CACAC U GAAUACC-3′ 7/42 =16.7% 7/38 = 18.4% (SEQ ID NO: 3) 3′-GU U CACAGUAGU G U G AC U UAU-5′(SEQ ID NO: 4) Column 1: The number refers to the nucleotide position ofthe 5′ base of the sense strand relative to the mouse ApoB mRNA sequenceXM_137955. Column 2: The numbers refer to the distribution of 2′OMechemical modifications in each strand. Column 3: 2′OMe-modifiednucleotides are indicated in bold and underlined. The siRNA duplex canalternatively or additionally comprise 2′-deoxy-2′-fluoro (2′F)nucleotides, 2′-deoxy nucleotides, 2′-O-(2-methoxyethyl) (MOE)nucleotides, and/or locked nucleic acid (LNA) nucleotides. Column 4: Thenumber and percentage of 2′OMe-modified nucleotides in the siRNA duplexare provided. Column 5: The number and percentage of modifiednucleotides in the double-stranded (DS) region of the siRNA duplex areprovided.

Regardless of the SNALP manufacturing process employed or the specificSNALP formulation screened, all SNALP formulations contained siApoB-8 asthe nucleic acid component, and all SNALP formulations contained thefollowing lipid components: the PEG-lipid conjugate PEG-cDMA(3-N-[(-Methoxypoly(ethyleneglycol)2000)carbamoyl]-1,2-dimyristyloxypropylamine); the cationic lipidDLinDMA (1,2-Dilinoleyloxy-3-(N,N-dimethyl)aminopropane); thephospholipid DSPC (1,2-Distearoyl-sn-glycero-3-phosphocholine; AvantiPolar Lipids; Alabaster, Ala.); and synthetic cholesterol (Sigma-AldrichCorp.; St. Louis, Mo.). The siRNAs were encapsulated into SNALP in oneof the following formulations (all of which are described in molarpercentages): (i) the “10:15” formulation: 10% PEG-cDMA; 15% DLinDMA;20% DSPC; and 55% cholesterol (ii) the “2:30” formulation: 2% PEG-C-DMA,30% DLinDMA, 20% DSPC, and 48% cholesterol; (iii) the “2:40”formulation: 1.4% PEG-cDMA; 57.1% DLinDMA; 7.1% DSPC; and 34.3%cholesterol; (iii) the “1:57” formulation: 1.4% PEG-cDMA; 57.1% DLinDMA;7.1% DSPC; and 34.3% cholesterol; or (iv) the “1:62” formulation: 1.5%PEG-cDMA, 62 mol DLinDMA, and 37% cholesterol. These variousformulations are summarized in tabular form in Table 3.

TABLE 3 Lipid Components of SNALP Formulations PEG2000-C-DMA DLinDMACholesterol DSPC (mol %) (mol %) (mol %) (mol %) 10:15  10 15 55 20 2:302 30 48 20 2:40 2 40 48 10 1:57 1.4 57 34 7 1:62 1.5 62 37 0

The lipid components and physical characteristics of the formulationsprepared by the Stepwise Dilution Method are summarized in Table 4. Thelipid:drug ratio is described in units of mg total lipid per mg nucleicacid. Mean particle size and polydispersity were measured on a MalvernInstruments Zetasizer. Encapsulation of nucleic acid was measured usinga Ribogreen assay essentially as described in Heyes et al., Journal ofControlled Release, 107:276-287 (2005).

TABLE 4 Characterization of Formulations Prepared by Stepwise DilutionMethod (SDM) Particle size Initial Final Formulation (nm)/Poly-Encapsulation Encapsulation Final (L/D) dispersity (%) (%) L/D 10:15  93(0.12) 87 95 15.8 (L/D 16.4) 2:30  95 (0.10) 96 96 10.2 (L/D 12.9) 2:40114 (0.14) 97 97 10.9 (L/D 12.5) 1:57 116 (0.06) 99 97 7.4 (L/D 9) 1:62108 (0.09) 100 96 6.4 (L/D 8)

The particle size ranged from 93-116 nm, and the polydispersity valuesranged from 0.06-0.14. Moreover, with the exception of the 10:15formulation, all formulations had high initial encapsulation >95%.

The lipid components and physical characteristics of the formulationsprepared by the Direct Dilution Method (DDM) are summarized in Table 5.As with Table 4, the lipid:drug ratio is described in units of mg totallipid per mg nucleic acid. Similarly, the mean particle size andpolydispersity were measured on a Malvern Instruments Zetasizer, and theencapsulation of nucleic acid was measured using a Ribogreen assayessentially as described in Heyes et al., Journal of Controlled Release,107:276-287 (2005).

TABLE 5 Characterization of Formulations Prepared By Direct DilutionMethod (DDM) Particle size Initial Final Formulation (nm)/Poly-Encapsulation Encapsulation Final (L/D) dispersity (%) (%) L/D 10:15 55(0.09) 30 94 47.2 (L/D 16.4) 2:30 58 (0.07) 97 97 11.6 (L/D 12.9) 2:4065 (0.11) 95 97 12.3 (L/D 12.5) 1:57 78 (0.03) 93 96 8.5 (L/D 9) 1:62 78(0.04) 86 97 8.7 (L/D 8)

The particle size ranged from 55-78 nm, and the polydispersity valuesranged from 0.03-0.09. The 10:15 formulation had low encapsulation (30%)and high final L/D.

From Tables 4 and 5, it is apparent that SNALP prepared by DDM (DirectDilution Method) possess smaller sizes and polydispersity values thanthose prepared by SDM (stepwise dilution method). Initial encapsulationfor SDM formulations were generally higher than for DDM formulations,most likely due to the acidic blending conditions in SDM. Further, it isnoted that the 10:15 SNALP prepared by DDM was problematic, with only30% encapsulation and high final L/D ratio, an indication that thisformulation is not compatible with this process method.

Example 2 Further Characterization of SNALP Structure Using Cryo-TEM

Various SNALP formulations prepared by the Stepwise Dilution Method andthe Direct Dilution Method were further characterized byCryo-Transmission Electron Microscopy (“Cryo-TEM”). As illustrated inFIGS. 2A-2C, Cryo-TEM is a microscopy technique, whereby a beam ofelectrons is transmitted through an ultra-thin frozen specimen,interacting with the speciment as it passes through. An image is thenformed from the interaction of the electrons transmitted through thespecimen, and the image is magnified and focused onto an imaging device.

Coded samples were sent to Uppsala University for Cryo-TEM imagingaccording to the method described by Almgren, M., et al., Colloid Surf.A 174, 3-21 (2000). Breifly, the samples were incubated at 25° C. for20-30 minutes before preparation. The climate chamber conditions were:25° C., with >98% relative humidity. 0.5 μL of sample solution wasdeposited on copper grid with perforated polymer film. Excess solutionwas removed by blotting and the sample was vitrified in liquid ethane.Images at 100,000× total magnification were captured. Diametrical sizeof particles were calculated by number averaging (Scale Bars all=100nm).

For all SNALP formulations analyzed, whether made by the SDM or the DDM,the resulting SNALP particles were categorized using the followingcriteria: (a) Non-lamellar particles: dense particles with no visiblelamellar structures (e.g., no bilayers); and (b) Lamellar particles:particles possessing bilayer structures, including those with multiplecompartments, LUVs and MLVs. It is noted that large structures withnon-spherical, irregular shapes were not included as these weresuspected to be artefacts. All SNALP samples analyzed contained 15 mg/mLtotal lipid and all samples were analyzed undiluted.

Table 6 sets forth the SNALP formulations prepared by the StepwiseDilution Method (SDM) that were analyzed by Cryo-TEM.

TABLE 6 SNALP Formulations Prepared by SDM and Analyzed by Cryo-TEM LotNo. Composition 284-050610-1 10:15  284-050610-2 2:30 284-050610-4 1:57284-050610-5 1:62

FIG. 3 sets forth representative Cryo-TEM data for the siApoB-8 10:15SNALP formulation prepared by the Stepwise Dilution Method. It was foundthat spherical non-lamellar particles were more abundant (n=594) thanlamellar particles (n=173). The particles varied in size, with rarelarge structures observed, which were thought to be artefacts.

FIG. 4 sets forth representative Cryo-TEM data for the siApoB-8 2:30SNALP formulation prepared by the Stepwise Dilution Method. This 2:30SNALP formulation had similar heterogeneous particle morphology to thatof the 10:15 formulation. It was found that non-lamellar particles weremore abundant (n=665) than lamellar particles (n=67). The particlesvaried in size, with some non-spherical particles being observed.

FIG. 5 sets forth representative Cryo-TEM data for the siApoB-8 1:57formulation prepared by the Stepwise Dilution Method. It was found thatpredominantly (>95%) non-lamellar particles were present (325 of 341);however, approximately half of the lamellar structure possessnon-lamellar compartments (see, examples marked with arrows in FIG. 5).It is clear from the Cryo-TEM data that the the particles size is morehomogeneous, with no large structures being found.

FIG. 6 sets forth representative Cryo-TEM data for the siApoB-8 1:62formulation prepared by the Stepwise Dilution Method. The 1:62 SNALPformulation has similar particle morphology to the 1:57 formulationwith >98% of the particles being non-lamellar particles (313 of 317).Similar to the 1:57 formulation, particle size is more homogeneous, withlarge particles rarely being found. Further, with the 1:62 formulation,fewer particles with dual compartments were present compared to the 1:57formulation (see, examples marked with arrows in FIG. 6).

FIG. 7 illustrates the presence of lamellar particles in the 10:15, the2:30, the 1:57 and the 1:62 SNALP formulations prepared using theStepwise Dilution Method. From FIG. 7, it is apparent that using theStepwise Dilution Methodology, an increase in the molar percentage ofcationic lipid in the SNALP formulation decreases the incidence oflamellar particles.

Table 7 sets forth the SNALP formulations prepared by the DirectDilution Method (DDM) that were analyzed by Cryo-TEM.

TABLE 7 SNALP Formulations Prepared by DDM and Analyzed by Cryo-TEM LotNo. Composition 280-060710-3 2:30 280-060710-2 2:40 280-060710-4 1:57280-060710-5 1:62

FIG. 8 sets forth representative Cryo-TEM data for the siApoB-8 2:30formulation prepared by the Direct Dilution Method. As seen in FIG. 8,the vast majority of particles in the 2:30 SNALP formulation arenon-lamellar (1386 of 1400).

FIG. 9 sets forth representative Cryo-TEM data for the siApoB-8 2:40formulation prepared by the Direct Dilution Method. As with the 2:30formulation, the vast majority of particles in the 2:40 SNALPformulation are non-lamellar (1191 of 1201 (or greater than 99% of theparticles)).

FIG. 10 sets forth representative Cryo-TEM data for the siApoB-8 1:57formulation prepared by the Direct Dilution Method. As with the 2:30 and2:40 formulations, the vast majority of particles in the 1:57 SNALPformulation are non-lamellar, with lamellar particles being very rare (2of 696). Advantageously, the particle size distribution is veryhomogeneous, with very low polydispersity.

FIG. 11 sets forth representative Cryo-TEM data for the siApoB-8 1:62formulation prepared by the Direct Dilution Method. As with the 2:30,the 2:40 and the 1:57 formulations, the vast majority of particles inthe 1:62 SNALP formulation are non-lamellar, with lamellar particlesbeing very rare (2 of 709). Advantageously, and similar to the 1:57formulation, particle size distribution is very homogeneous in the 1:62formulation, with very low polydispersity.

FIG. 12 illustrates the presence of lamellar particles in the 2:30, the2:40, the 1:57 and the 1:62 SNALP formulations prepared using the DirectDilution Method. From FIG. 12, it is seen that the Direct DilutionMethod favors non-lamellar particle formation. Moreover, it is seen thatincreasing the cationic lipid content has a minor effect on reducing theincidence of lamellar particles.

Based on the Cryo-TEM data, it is clear that the Stepwise DilutionMethod (SMD) produces SNALP with a higher proportion of lamellarparticles compared to SNALP prepared using the Direct Dilution Method.The 1:57 and 1:62 SNALP formulations produce particles with veryhomogeneous morphologies. With these formulations, the SNALP formationmethod has a large impact on particle size, but only a minor effect onparticle morphology (since with both the SDM and the DDM, the vastmajority of particles were non-lamellar).

For certain other formulations (e.g., the 10:15 and the 2:30 SNALPformulations), particle morphology is, in fact, dependent on formationmethod (SDM vs. DDM). Interestingly, it was found that the DirectDilution Method is unable to produce the 10:15 SNALP formulation withacceptable siRNA encapsulation.

This Cryo-TEM data clearly demonstrates that a novel, non-liposomallipid nanoparticle can be prepared using the Direct Dilution Methoddescribed herein. The resulting SNALP particles contain pH titratableaminolipids, are charge-free at physiological pH and encapsulate polarnucleic acid. Importantly, the Direct Dilution Method yielded smallerparticle sizes and smaller polydispersity than those prepared by theSDM. It is thought that tightening the particle size distribution usingthe Direct Dilution Method helps to reduce the incidence of lamellarparticles.

Example 3 Characterization of 7:54 SNALP Formulation Using Cryo-TEM

In this example, the 7:54 formulation as well as variations of the 7:54formulation were analyzed using methods similar to those set forth inExamples 1 and 2. All of the 7:54 SNALP formulations were prepared withan siRNA targeting polo-like kinase 1 (PLK-1) (Genbank Accession No.NM_005030) as the nucleic acid component. The PLK-1 siRNA sequence usedin this study is provided in Table 8.

TABLE 8 % 2′OMe- % Modified siRNA PLK-1 siRNA Sequence Modifiedin DS Region PLK1424 2/6 5′-AGA U CACCC U CCU U AAA U A U U-3′ 9/42 =21.4% 7/38 = 18.4% (SEQ ID NO: 1) 3′-C U UC U A G UGGGAG G AAUUUAU-5′(SEQ ID NO: 2) Column 1: The number after “PLK” refers to the nucleotideposition of the 5′ base of the sense strand relative to the start codon(ATG) of the human PLK-1 mRNA sequence NM_005030. Column 2: 2′OMenucleotides are indicated in bold and underlined. The 3′-overhangs onone or both strands of the siRNA molecule may alternatively comprise 1-4deoxythymidine (dT) nucleotides, 1-4 modified and/or unmodified uridine(U) ribonucleotides, or 1-2 additional ribonucleotides havingcomplementarity to the target sequence or the complementary strandthereof. Column 3: The number and percentage of 2′OMe-modifiednucleotides in the siRNA molecule are provided. Column 4: The number andpercentage of modified nucleotides in the double- stranded (DS) regionof the siRNA molecule are provided.

The lipid components and physical characteristics of the various 7:54formulations prepared by the Direct Dilution Method are summarized inTable 9. Mean particle size and polydispersity were measured on aMalvern Instruments Zetasizer. Encapsulation of nucleic acid wasmeasured using a Ribogreen assay essentially as described in Heyes etal., Journal of Controlled Release, 107:276-287 (2005).

TABLE 9 Characterization of 7:54 Formulations Prepared by DirectDilution Method Composition (mol %) Finished Product PEG750-C- SNALPDMA|DLinDMA Zavg Encaps Lot Description | Chol | DPPC (nm) Poly (%) 121-7:54 6.76|54.06|32.43|6.75 79.01 0.075 93 050709-1 121- 7:54 − 25%5.15|54.98|32.99|6.87 89.03 0.037 93 050709-2 PEG (5:55) 121- 7:54 + 50%9.80|52.29|31.37|6.5 67.57 0.085 94 050709-4 PEG (10:52) “Zavg” = mediandiameter of particle; “Poly” = polydispersity; “Encaps” = encapsulationefficiency.

The various 7:54 formulations were further characterized byCryo-Transmission Electron Microscopy (“Cryo-TEM”) as described inExample 2. Coded samples were sent to Uppsala University for Cryo-TEMimaging according to the method described by Almgren, M., et al.,Colloid Surf. A 174, 3-21 (2000). Breifly, the samples were incubated at25° C. for 20-30 minutes before preparation. The climate chamberconditions were: 25° C., with >98% relative humidity. 0.5 μL of samplesolution was deposited on copper grid with perforated polymer film.Excess solution was removed by blotting and the sample was vitrified inliquid ethane. Images at 100,000× total magnification were captured.Diametrical size of particles were calculated by number averaging (ScaleBars all=100 nm).

FIG. 13 sets forth representative Cryo-TEM data for the PLK-1 7:54formulation prepared by the Direct Dilution Method. As with the 1:57 and1:62 formulations, the vast majority of particles in the 7:54 SNALPformulation are non-lamellar, with lamellar particles being very rare.Also, similar to the 1:57 and 1:62 formulations, particle sizedistribution is very homogeneous in the 7:54 formulation, with very lowpolydispersity.

FIGS. 14 and 15 also set forth representative Cryo-TEM data for the 7:54PEG₇₅₀-C-DMA (−25% PEG) PLK SNALP and the 7:54 PEG750-C-DMA (+50% PEG)PLK SNALP formulations, respectively, prepared by the Direct DilutionMethod. As with the 7:54 SNALP formulation, the vast majority ofparticles in these variations of the 7:54 SNALP formulation arenon-lamellar, with lamellar particles being very rare. Also, similar tothe 7:54 formulation, particle size distribution is very homogeneous inthese variations of the 7:54 SNALP formulations, with very lowpolydispersity.

Example 4 Comparison of the Silencing Activity of ApoB siRNA Formulatedas 2:30 SNALP and 2:40 SNALP

SNALP formulations were prepared with the ApoB siRNA set forth in Table2. The 2:30 SNALP formulation used in this study is lipid composition2:30:20:48 as described in molar percentages of PEG-C-DMA, DLinDMA,DSPC, and cholesterol (in that order). The 2:40 SNALP formulation usedin this study is lipid composition 2:40:10:48 as described in molarpercentages of PEG-C-DMA, DLinDMA, DPPC, and cholesterol (in thatorder).

BALB/c mice (female, 4 weeks old) were obtained from Harlan Labs. Afteran acclimation period (of at least 7 days), animals were administered asingle dose of SNALP by intravenous (IV) injection in the lateral tailvein. The dose was 5 mg encapsulated siRNA per kg body weight. As anegative control, one group of animals was given IV injections ofphosphate buffered saline (PBS) vehicle. 96 h after the treatment,animals were euthanized and liver tissue was collected in RNAlater.

Liver tissues were analyzed for ApoB mRNA levels normalized againstglyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels using theQuantiGene assay (Panomics; Fremont, Calif.) essentially as described inJudge et al., Molecular Therapy, 13:494 (2006).

FIG. 16 shows that the 2:40 SNALP containing ApoB 10048 U2/2 G1/2 siRNAwas about 3 times as efficacious as the 2:30 SNALP in mediating ApoBgene silencing in mouse liver.

Example 5 ApoB siRNA Formulated as 1:57 SNALP have Potent SilencingActivity In Vivo

SNALP formulations were prepared with the ApoB siRNA set forth in Table2. The lipid components and physical characteristics of the formulationsare summarized in Table 10. The lipid:drug ratio is described in unitsof mg total lipid per mg nucleic acid. Mean particle size andpolydispersity were measured on a Malvern Instruments Zetasizer.Encapsulation of nucleic acid was measured using a Ribogreen assayessentially as described in Heyes et al., Journal of Controlled Release,107:276-287 (2005).

TABLE 10 Characteristics of the SNALP formulations used in this study.SNALP siRNA Particle Size % (L:D ratio) Payload (Polydispersity)Encapsulation 2:30 (13) ApoB-10048 65 nm (0.16) 88 U2/2 G1/2 1:57 (9)ApoB-10048 74 nm (0.10) 89 U2/2 G1/2

The 2:30 SNALP formulation used in this study is lipid composition2:30:20:48 as described in molar percentages of PEG-C-DMA, DLinDMA,DSPC, and cholesterol (in that order). This formulation was prepared bysyringe press at an input lipid to drug (L:D) ratio (mg:mg) of 13:1.

The 1:57 SNALP formulation used in this study is lipid composition1.5:57.1:7:34.3 as described in molar percentages of PEG-C-DMA, DLinDMA,DPPC, and cholesterol (in that order). This formulation was prepared bysyringe press at an input lipid to drug (L:D) ratio (mg:mg) of 9:1.

BALB/c mice (female, 4 weeks old) were obtained from Harlan Labs. Afteran acclimation period (of at least 7 days), animals were administeredSNALP by intravenous (IV) injection in the lateral tail vein once dailyon Study Days 0, 1, 2, 3 & 4 for a total of 5 doses per animal. Dailydosage was either 1.0 (for 2:30 SNALP) or 0.1 (for 1:57 SNALP) mgencapsulated siRNA per kg body weight, corresponding to 10 ml/kg(rounded to the nearest 10 μl. As a negative control, one group ofanimals was given IV injections of phosphate buffered saline (PBS)vehicle. On Study Day 7, 72 h after the last treatment, animals wereeuthanized and liver tissue was collected in RNAlater.

Liver tissues were analyzed for ApoB mRNA levels normalized againstglyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels using theQuantiGene assay (Panomics; Fremont, Calif.) essentially as described inJudge et al., Molecular Therapy, 13:494 (2006).

FIG. 17 shows that the 1:57 SNALP containing ApoB 10048 U2/2 G1/2 siRNAwas more efficacious than the 2:30 SNALP in mediating ApoB genesilencing in mouse liver at a 10-fold lower dose.

Example 6 Comparison of the Silencing Activity of ApoB siRNA Formulatedas 2:40 SNALP and 1:57 SNALP

SNALP formulations were prepared with the ApoB siRNA set forth in Table2, i.e., siApoB-8. The 2:40 SNALP formulation used in this study islipid composition 2:40:10:48 as described in molar percentages ofPEG-C-DMA, DLinDMA, DPPC, and cholesterol (in that order). The 1:57SNALP formulation used in this study is lipid composition1.5:57.1:7:34.3 as described in molar percentages of PEG-C-DMA, DLinDMA,DPPC, and cholesterol (in that order).

BALB/c mice (female, 4 weeks old) were obtained from Harlan Labs. Afteran acclimation period (of at least 7 days), animals were administered asingle dose of SNALP by intravenous (IV) injection in the lateral tailvein. Dosage was either 0.5 or 0.75 (for the 2:40 SNALP), or 0.10, 0.25,0.5 or 0.75 (for the 1:57 SNALP) mg encapsulated siRNA per kg bodyweight, corresponding to 10 ml/kg (rounded to the nearest 10 μl). As anegative control, one group of animals was given IV injections ofphosphate buffered saline (PBS) vehicle. 48 h after the treatment,animals were euthanized and liver tissue was collected in RNAlater.

Liver tissues were analyzed for ApoB mRNA levels normalized againstglyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels using theQuantiGene assay (Panomics; Fremont, Calif.) essentially as described inJudge et al., Molecular Therapy, 13:494 (2006).

FIG. 18 shows that the 1:57 SNALP containing ApoB 10048 U2/2 G1/2 siRNAwas as efficacious as the 2:40 SNALP in mediating ApoB gene silencing inmouse liver at a 7.5-fold lower dose, i.e., 64% knockdown at a 0.75mg/kg dose of 2:40 SNALP versus 63% knockdown at a 0.10 mg/kg dose of1:57 SNALP.

Example 7 Activity of the 7:54 DLinDMA SNALP Formulation in Normal LiverVersus Liver Tumors

7:54 or 1:57 DLinDMA SNALP formulations were prepared with PLK-1 (Table8) or ApoB (Table 2) siRNA as the nucleic acid component. Mice withnormal livers were administered either phosphate buffered saline (PBS),ApoB 1:57 DLinDMA SNALP, or ApoB 7:54 DLinDMA SNALP by intravenous (IV)injection via the lateral tail vein. Mice with established Hep3Bintrahepatic tumors were administered either PBS, PLK-1 1:57 DLinDMASNALP, or PLK-1 7:54 DLinDMA SNALP by IV injection via the lateral tailvein.

FIG. 19 shows that the 1:57 DLinDMA SNALP formulation was capable ofsilencing ApoB expression in normal liver tissue and PLK-1 expression inHep3B liver tumors, while the 7:54 DLinDMA SNALP formulation displayedenhanced silencing activity in liver tumors compared to normal livertissue. As such, this example demonstrates that the 7:54 DLinDMA SNALPformulation preferentially targets tumor cells compared to the normalliver, whereas the 1:57 DLinDMA SNALP formulation preferentially targetsnormal liver cells compared to solid tumors.

This example further demonstrates that the 7:54 DLinDMA SNALPformulation may help limit PLK-1 silencing in proliferating hepatocytes(e.g., diseased liver state). Hepatocytes in the healthy liver aretypically non-dividing and therefore do not express PLK-1. However, indiseased states (e.g., in a cancerous liver), normal hepatocytes aremore proliferative (as they attempt to repair the damage) and thereforeexpress PLK-1. Use of the PLK-1 7:54 DLinDMA SNALP formulation avoidsthe undesired targeting of normal proliferating hepatocytes, therebylimiting PLK-1 silencing in these cells. As a result, there is areduction or abrogation in the death of these healthy hepatocytes, whilePLK-1 expression is effectively silenced in tumor cells.

Example 8 Activity of the 7:54 DLinDMA SNALP Formulation in TumorsOutside of the Liver

7:54 or 1:57 DLinDMA SNALP formulations were prepared with PLK-1 siRNAas the nucleic acid component (Table 8). Mice with established Hep3Bsubcutaneous (SC) tumors were administered either PBS (Control), PLK-11:57 DLinDMA SNALP, or PLK-1 7:54 DLinDMA SNALP by IV injection via thelateral tail vein at a dose of 6×3 mg/kg SNALP twice weekly for 3 weeks(Days 17, 20, 24, 27, 31, 34). FIG. 20 shows that while multiple dosesof PLK-1 1:57 DLinDMA SNALP were effective at inducing the regression ofestablished SC Hep3B tumors compared to control mice, multiple doses ofPLK-1 7:54 DLinDMA SNALP were more effective at inducing the regressionof these SC solid tumors compared to the PLK-1 1:57 DLinDMA SNALPformulation.

Thus, this study shows that the 7:54 DLinDMA SNALP formulation displaysincreased potency in SC tumors and can be used to preferentially targettumors outside of the liver.

Example 9 Synthesis of MC3

MC3 (Compound 1) having the structure shown below was synthesized asdescribed in Scheme 1 below.

STEP 1: Magnesium bromide etherate (34 g, 110 mmol) and a stir bar wereadded to a 2000 mL round bottom flask. The flask was sealed and flushedwith nitrogen. Anhydrous diethyl ether (400 mL) was added via canulla. Asolution of linolenyl mesylate (20 g, 58 mmol) in anhydrous ether (300mL) was then added, and the suspension stirred overnight. The suspensionwas poured into 500 mL of chilled water and transferred to a 2000-mLseparating funnel. After shaking, the organic phase was separated. Theaqueous phase was then extracted with ether (2×250 mL) and all etherphases combined. The ether phase was washed with water (2×250 mL), brine(250 mL) and dried over anhydrous Mg₂SO₄. The solution was filtered,concentrated and purified by flash chromatography. Final yield 18.9 g,99%.

STEP 2: A 1 liter RBF was charged with magnesium turnings (11.1 g, 463mmol), anhydrous THF (65 mL) and stir-bar and flushed with nitrogen. Ina separate flask, a solution of linoleyl bromide (140 g, 425 mL) inanhydrous THF (150 mL) was prepared, and 20 mL of this solution added tothe magnesium. When most of the heat had dissipated, the remainder ofthe bromide was added over a period of 15 minutes. Temperature was thenmaintained at 45° C. for 4 h. The reaction was then cooled (0° C.).Using a dropping funnel, a solution of ethyl formate (32.4 g, 438 mmol)in anhydrous THF (150 mL) was added over a 40 minute period. Thereaction was stirred overnight at room temperature. The reaction wascooled to −15° C. and 5N HCl (185 mL) added slowly. The mixture wastransferred to a 2 L separating funnel separated. Water (150 mL) andhexane (150 mL) were added, the mixture washed, and again the aqueousremoved. The organic was washed a final time with water (150 mL) andthen concentrated to a yellow oil. The yellow oil was stirred with amixture of EtOH (210 mL), water (30 mL) and KOH (15.8 g) for 1.5 h atroom temp. The EtOH was evaporated and the residue treated with hexane(50 mL). 5N HCl (200 mL) was added via dropping funnel. The organic waswashed with water (2×50 mL) evaporated, dried and purified bychromatography (0-5% EtOAc in hexane, 91 g, 81%).

STEP 3: Dilinoleylmethanol (7.8 g, 14.9 mmol), dimethylaminobutyric acidhydrochloride (2.99 g, 17.8 mmol) and a stir bar were added to 500 mLRBF. The flask was flushed with nitrogen and anh. DCM (120 mL) added,followed by EDCI (3.6 g, 18.8 mmol), DIPEA (6.3 mL, 36.3 mmol) and DMAP(450 mg, 3.69 mmol). The reaction was stirred overnight. The reactionwas diluted with DCM (300 mL) and washed with sat. NaHCO₃ (200 mL),water (300 mL) and sat. NaCL (200 mL). Each aq. wash was extracted oncewith DCM (50 mL). Organics were combined, dried (MgSO₄) and concentratedto yield a yellow oil with some crystalline matter. This was purified bychromatography (0-2% MeOH in CHCl₃) to yield Lin-MC3 as a pale yellowoil (9.0 g, 14.1 mmol, 95%).

Example 10 Synthesis of LenMC3 and CP-LenMC3

LenMC3 (Compound 4) and CP-LenMC3 (Compound 5) having the structuresshown below were synthesized as described in Scheme 2 below. LenMC3 isalso known as linolenyl-MC3 and DLen-MC3. CP-LenMC3 is also known asCP-linolenyl-MC3 and CP-DLen-MC3.

Synthesis of Linolenyl Bromide (Compound 2)

Magnesium bromide etherate (34 g, 110 mmol) and a stir bar were added toa 2000 mL round bottom flask. The flask was sealed and flushed withnitrogen. Anhydrous diethyl ether (400 mL) was added via canulla. Asolution of linolenyl mesylate (20 g, 58 mmol) in anhydrous ether (300mL) was then added, and the suspension stirred overnight. The suspensionwas poured into 500 mL of chilled water and transferred to a 2000-mLseparating funnel. After shaking, the organic phase was separated. Theaqueous phase was then extracted with ether (2×250 mL) and all etherphases combined. The ether phase was washed with water (2×250 mL), brine(250 mL) and dried over anhydrous Mg₂SO₄. The solution was filtered,concentrated and purified by flash chromatography. Final yield 19.1 g,100%.

Synthesis of Dilinolenyl Methanol (Compound 3)

Magnesium turnings (2.1 g, 87 mmol), 5 crystals of iodine and a stirbarwere added to a 1000 mL round-bottom flask. The flask was flushed withnitrogen and a solution of linolenyl bromide (Compound 2) (19.1 g, 58mmol) in anhydrous diethyl ether (500 mL) added via cannula. The mixtureturned cloudy and was refluxed overnight. The mixture was cooled to RTand ethyl formate (4.66 mL, 58 mmol) added via syringe. The addition wasmade dropwise, directly into the reaction mixture, and the cloudysuspension again stirred overnight. During this time the reaction turnedbright yellow. The R.M. was transferred to a 2000-mL sep. funnel withether (50 mL), and washed with 10% H₂SO₄ (200 mL), water (2×200 mL) andbrine (200 mL). The organic was dried over anhydrous MgSO₄, filtered andconcentrated. Crude yield was 14.5 g. TLC indicated that majority ofproduct was the methyl formate, which was purified by columnchromatography. The purified formate (9.3 g, 16.7 mmol) was transferredto a 1000 mL round bottom flask and EtOH (600 mL) and a stirbar added.With stirring, water (25 mL—forming ˜5% aqueous solution) was slowlyadded, followed by KOH (2.0 g, 35.7 mmol). After 1 hour, the solutionhad turned pale yellow. TLC indicated reaction had gone to completion.The solution was concentrated by rotovap to 50% of its volume and thenpoured into 200 mL of 5% HCl. The aqueous phase was extracted with ether(3×200 mL). The ether fractions were combined and washed with water(3×200 mL), dried (MgSO₄) and concentrated to yield 8.9 g of dilinolenylmethanol (16.8 mmol, 58%).

Synthesis of Len-MC3 (Compound 4)

Dilinolenyl methanol (Compound 3) (2.5 g, 4.76 mmol),dimethylaminobutyric acid hydrochloride (970 mg, 5.77 mmol) and a stirbar were added to 100 mL RBF. The flask was flushed with nitrogen andanhydrous DCM (40 mL) added, followed by EDCI (FW 191.7, 1.2 g, 6.26mmol), DIPEA (2.1 mL, 12.1 mmol) and DMAP (150 mg, 1.23 mmol). Thereaction was stirred overnight, whereupon TLC indicated >80% conversion.Reaction was diluted with DCM (100 mL) and washed with sat. NaHCO₃ (100mL), water (200 mL) and sat. NaCL (100 mL). Aqueous washes were combinedand extracted with DCM (2×50 mL). Organics were then combined, dried(MgSO₄) and concentrated to yield a yellow oil with some crystallinematter. This was purified by chromatography to yield Len-MC3 as a paleyellow oil (2.3 g, 3.6 mmol, 76%).

Synthesis of CP-LenMC3 (Compound 5)

To a 250 mL RBF was added Len-MC3 (Compound 4) (1.1 g, 1.72 mmol), astirbar and anhydrous DCM (40 mL). The flask was flushed with N₂ andcooled to 0° C., then a 1M solution of diethylzinc in hexanes added (30mL, 30 mmol). The solution was stirred for 1 hour at 0° C., thendiiodomethane (2.4 mL 30 mmol) added and the reaction stirred overnightat RT. The reaction mixture was concentrated and then redissolved inEtOAc (50 mL). The EtOAc was washed successively with 5% HCl (2×50 mL),water (50 mL), NaHCO₃ (50 mL), water (50 mL), and brine (50 mL). Theaqueous washes were combined and extracted with DCM (2×50 mL). Allorganics were combined, dried and concentrated to yield crudeCP-Len-MC3. ¹H-NMR indicated some olefins still to be present, so thecompound was treated again, using the same procedures and amountsoutlined above. This time, after chromatography, ¹H-NMR indicated totalconversion of the olefins. Final yield 1.0 g, 1.39 mmol, 81%.

Example 11 Synthesis of γ-LenMC3 and CP-γ-LenMC3

γ-LenMC3 (Compound 8) and CP-γ-LenMC3 (Compound 9) having the structuresshown below were synthesized as described in Scheme 3 below. γ-LenMC3 isalso known as γlinolenyl-MC3, γDLen-MC3, and D-γ-Len-MC3. CP-γ-LenMC3 isalso known as CP-γlinolenyl-MC3, CP-γDLen-MC3, and CP-D-γ-Len-MC3.

Synthesis of γ-Linolenyl Bromide (Compound 6)

Magnesium bromide etherate (34 g, 110 mmol) and a stir bar were added toa 2000 mL round bottom flask. The flask was sealed and flushed withnitrogen. Anhydrous diethyl ether (400 mL) was added via canulla. Asolution of γ-linolenyl mesylate (20 g, 58 mmol) in anhydrous ether (300mL) was then added, and the suspension stirred overnight. The suspensionwas poured into 500 mL of chilled water and transferred to a 2000-mLseparating funnel. After shaking, the organic phase was separated. Theaqueous phase was then extracted with ether (2×250 mL) and all etherphases combined. The ether phase was washed with water (2×250 mL), brine(250 mL) and dried over anhydrous Mg₂SO₄. The solution was filtered,concentrated and purified by flash chromatography. Final yield 18.9 g,99%.

Synthesis of Di-γ-Linolenyl Methanol (Compound 7)

Magnesium turnings (2.1 g, 87 mmol), 5 crystals of iodine and a stirbarwere added to a 1000 mL round-bottom flask. The flask was flushed withnitrogen and a solution of γ-linolenyl bromide (Compound 6) (18.9 g, 57mmol) in anhydrous diethyl ether (500 mL) added via cannula. The mixtureturned cloudy and was refluxed overnight. The mixture was cooled to RTand ethyl formate (4.66 mL, 58 mmol) added dropwise. The suspension wasstirred overnight, turning bright yellow. The R.M. was transferred to a2000-mL sep. funnel with ether (50 mL), and washed with 10% sulphuricacid (200 mL), water (2×200 mL) and brine (200 mL). The organic wasdried over anhydrous MgSO₄, filtered and concentrated. Crude yield was14.5 g. TLC indicated that majority of product was the methyl formate,which was purified by column chromatography. The purified formate wastransferred to a 1000 mL round bottom flask and EtOH (600 mL) and astirbar added. With stirring, water (25 mL—forming ˜5% aqueous solution)was slowly added, followed by KOH (2.0 g, 35.7 mmol). After 1 hour,solution had turned pale yellow. TLC indicated reaction had gone tocompletion. The solution was concentrated by rotovap to 50% of itsvolume and then poured into 200 mL of 5% HCl. The aqueous phase wasextracted with ether (3×200 mL). The ether fractions were combined andwashed with water (3×200 mL), dried (MgSO₄) and concentrated to yield8.8 g of di-γ-linolenyl methanol (16.8 mmol, 58%).

Synthesis of γ-LenMC3 (Compound 8)

Di-γ-linolenyl methanol (Compound 7) (2.5 g, 4.76 mmol),dimethylaminobutyric acid hydrochloride (970 mg, 5.77 mmol) and a stirbar were added to 100 mL RBF. The flask was flushed with nitrogen andanhydrous DCM (40 mL) added, followed by EDCI (1.2 g, 6.26 mmol), DIPEA(2.1 mL, 12.1 mmol) and DMAP (150 mg, 1.23 mmol). The reaction wasstirred overnight. The reaction was diluted with DCM (100 mL) and washedwith sat. NaHCO₃ (100 mL), water (200 mL) and sat. NaCL (100 mL).Aqueous washes were combined and extracted with DCM (2×50 mL). Organicswere then combined, dried (MgSO₄) and concentrated to yield a yellowoil. This was purified by chromatography to yield γ-Len-MC3 as a paleyellow oil (2.6 g, 4.1 mmol, 86%).

Synthesis of CP-γ-LenMC3 (Compound 9)

To a 250 mL RBF was added γ-LenMC3 (Compound 8) (1.28 g, 2.0 mmol), astirbar and anhydrous DCM (40 mL). The flask was flushed with N₂ andcooled to 0° C., then a 1M solution of diethylzinc in hexanes added (30mL, 30 mmol, ˜5 equivalents per olefin). The solution was stirred for 1hour at 0° C., then diiodomethane (2.4 mL 50 mmol) added and thereaction stirred overnight at RT. The reaction mixture was concentratedand then redissolved in EtOAc (50 mL). The EtOAc was washed successivelywith 5% HCl (2×50 mL), water (50 mL), NaHCO₃ (50 mL), water (50 mL), andbrine (50 mL). The aqueous washes were combined and extracted with DCM(2×50 mL). All organics were combined, dried and concentrated to yieldcrude CP-γ-LenMC3. ¹H-NMR indicated some olefins still to be present, sothe compound was treated again, using the same procedures and amountsoutlined above. This time ¹H-NMR indicated total conversion of theolefins. Final yield after chromatography was 1.3 g, 1.8 mmol, 90%.

Example 12 Synthesis of MC3MC

MC3MC (Compound 10) having the structure shown below was synthesized asdescribed in Schemes 4 and 5 below.

A 50 mL round bottom flask was charged with dilinoleyl methanol (3.06 g,5.78 mmol) and a stir bar and flushed with nitrogen. Anhydrous DCM (30mL) was added, followed by diphosgene (1.75 mL, 14.46 mmol, 2.5 eqv.).The reaction was stirred overnight and then concentrated by rotovap andpurified by chromatography. This yielded the product as a colourless oil(2.6 g, 4.4 mmol, 76%).

A 50 mL r.b.f. containing the chloroformate (350 mg, 0.59 mmol) and astir bar was flushed with nitrogen and sealed. Anhydrous DCM (10 mL) andN,N,N′-trimethyl-1,3-propanediamine (580 mg, 5 mmol) were added and thereaction stirred for 4 h. TLC indicated the reaction to have gone tocompletion. The mixture was diluted to a volume of 40 mL with DCM andwashed with sat. NaHCO₃ (30 mL), water (30 mL) and brine (30 mL). Theaqueous phases were combined and extracted once with DCM (20 mL).Organics were then combined, dried over MgSO₄, and concentrated byrotovap. Purification yielded the product as a pale oil, 350 mg, 0.52mmol, 89%.

Example 13 Synthesis of MC2MC

MC2MC (Compound 11) having the structure shown below was synthesized asdescribed in Scheme 6 below.

A 50 mL round bottom flask containing the chloroformate (400 mg, 0.68mmol) and a stir bar was flushed with nitrogen and sealed. Anhydrous DCM(10 mL) and N,N,N′-trimethyl-1,2-ethanediamine (510 mg, 5 mmol) wereadded and the reaction stirred for overnight. TLC indicated the reactionto have gone to completion. The mixture was concentrated by rotovap andpurified by column chromatography to yield the product as a pale oil(350 mg, 0.53 mmol, 78%).

Example 14 Synthesis of MC2C

MC2C (Compound 12) having the structure shown below was synthesized asdescribed in Scheme 7 below.

A 50 mL round bottom flask containing the chloroformate (400 mg, 0.68mmol) and a stir bar was flushed with nitrogen and sealed. Anhydrous DCM(10 mL) and N,N,-dimethylethylenediamine (440 mg, 5 mmol) were added andthe reaction stirred for overnight. TLC indicated the reaction to havegone to completion. The mixture was concentrated by rotovap and purifiedby column chromatography to yield the product as a pale yellow oil (350mg, 0.54 mmol, 80%).

Example 15 Synthesis of MC3 Ether

MC3 Ether (Compound 13) having the structure shown below was synthesizedas described in Scheme 8 below.

A 50 mL RBF with stir-bar was flushed with nitrogen and anhydrous DCM (4mL). Triflic anhydride (0.7 g, 420 μL, 2.5 mmol) was added and the flaskcooled to −15° C. Anhydrous pyridine (198 mg, 202 μL, 2.5 mmol) wasslowly added, causing fuming and a white precipitate to form. A solutionof dlinoleyl methanol (1.06 g, 2 mmol) in anhydrous DCM (2 mL) was addedslowly over a period of 2 minutes. After stirring for 2 h at ˜−15° C.the reaction was off-white in color. TLC showed triflate formation andwater (2 mL) was added to quench the reaction. DCM (10 mL) was added andthe mixture washed with water (2×20 mL), dried (MgSO₄), filtered andtransferred to a 25 mL round bottom flask. Proton Sponge (1.07 g, 5mmol, min 2.5 eqv.), dimethylaminopropanol (515 mg, 5 mmol, min. 2.5eqv) and a stir bar added and the vessel flushed with nitrogen, fittedwith a condenser and refluxed for 48 h. Water (10 mL) was added, andafter stirring vigorously for several minutes, separated in a 30 mL sepfunnel. The organic was washed again with water (10 mL), dried overMgSO₄, concentrated and purified by chromatography (MeOH/CHCl₃) to yieldthe product as a pale yellow oil (400 mg, 33%).

Alternatively, MC3 Ether (Compound 13) was synthesized starting fromdilinoleyl methanol (DLinMeOH) as follows:

Synthesis of Compound 14

A 50 mL RBF with stir-bar was flushed with nitrogen, and DLinMeOH (1060mg, 2 mmol), TEA (6 mmol, 834 μL) and anh. DCM (20 mL) added. Flask wascooled to 0° C. and either MsCl (6 mmol) added. Reaction was stirredovernight. Reaction was diluted to 70 mL with DCM, washed with sat.NaHCO₃ (2×50 mL) and sat. NaCl (50 mL), dried (MgSO₄), filtered,concentrated and purified by column chromatography (1-4% EtOAc inhexane). Yield 900 mg, 75%.

Synthesis of MC3 Ether

A 50 mL RBF with stir-bar were flushed with nitrogen, and NaH (220 mg, 9mmol), dimethylaminopropanol (927 mg, 1.06 mL, 9 mmol) and anh. benzene(10 mL) added. After effervescence subsided, Compound 14 (440 mg, 0.75mmol) was added and RM refluxed overnight at 90° C. TLC indicated 30-50%product formation. RM was refluxed a second night, but TLC did notappear to indicate further reaction. The reaction was diluted to 40 mLwith benzene, and quenched with ethanol (25 mL). It was then washed withwater (40 mL), dried and concentrated. The crude product was purified toyield product as a pale yellow oil, 157 mg, 33%.

Example 16 Synthesis of MC4 Ether

MC4 Ether (Compound 15) having the structure shown below was synthesizedas described below.

A 50 mL RBF with stir-bar were flushed with nitrogen, and NaH (220 mg, 9mmol), dimethylaminobutanol (1.05 g, 9 mmol) and anh. benzene (10 mL)added. After effervescence subsided, Compound 14 (440 mg, 0.75 mmol) wasadded and RM refluxed overnight at 90° C. TLC indicated some productformation. The reaction was diluted to 40 mL with benzene, and quenchedwith ethanol (25 mL). It was then washed with water (40 mL), dried andconcentrated. The crude product was purified to yield product as a paleyellow oil, 145 mg, 31%.

Example 17 Synthesis of MC3 Amide

MC3 Amide (Compound 16) having the structure shown below was synthesizedas described in Schemes 9-11 below.

To a 500 mL RBF containing a solution of dilinoleyl methanol (10 g, 18.9mmol) in DCM (200 mL) was added pyridinium chlorochromate (12.24 g, 56.7mmol), anh. sodium carbonate (1.0 g, 9.5 mmol) and a stir bar. Theresulting suspension was stirred under nitrogen at RT for 3 h, afterwhich time TLC indicated all SM to have been consumed. Ether (300 mL)was then added to the mixture and the resulting brown suspensionfiltered through a pad of silica (300 mL), washing the pad with ether(3×100 mL). The ether phases were combined, concentrated and purified toyield 9.0 g (17.1 mmol, 90%) of ketone.

To a solution of dilinoleyl ketone (1.0 g, 1.9 mmol) in 2M ammonia inethanol (5 mL) was added titanium(IV) isopropoxide (1.15 mL, 3.8 mmol).The solution was stirred under nitrogen at room temperature for 6 hoursthen sodium borohydride (110 mg, 3.8 mmol) was added. The solutioneffervesced for approximately 5 minutes, and then a colorlessprecipitate began to form. The solution was stirred for 16 hours at roomtemperature, quenched with 10% NH₄OH (25 mL) and diluted with ethylacetate (50 mL). The inorganic solids were filtered and the aqueousphase was washed with ethyl acetate (2×75 mL). The combine ethyl acetateextracts were washed with 2M HCl (2×50 mL), dried on magnesium sulfate,filtered and concentrated to dryness to afford the product as a paleyellow HCl salt (1.1 g, quantitative).

To a solution of dilinoleyl methylamine hydrochloride (1.1 g, 1.95mmol), BOP (1.1 g, 2.4 mmol) and 4-(dimethylamino)butanoic acidhydrochloride (402 mg, 2.4 mmol) in anhydrous DMF (20 mL) was addeddiisopropylethylamine (1.4 mL, 7.8 mmol). The solution was stirred for16 hours at room temperature. The solution was concentrated in vacuo todryness and dissolved in ethyl acetate (100 mL). The ethyl acetate waswashed with brine (3×50 mL), dried on magnesium sulfate, filtered andconcentrated in vacuo to dryness. The residue was purified by columnchromatography (1% to 2.5% MeOH in CHCl₃) to afford the product as anorange oil. Decolorization through a pad of silica gel (eluted with 50%hexanes ethyl acetate to 100% ethyl acetate) afforded the product as apale yellow oil (151 mg, 12%).

Example 18 Synthesis of Pan-MC3

Phytanyl-MC3 (“Pan-MC3”) (Compound 17) having the structure shown belowwas synthesized as described in Scheme 12 below.

Synthesis of Phytanyl Mesylate

To a solution of phytanol (14.98 g, 50.2 mmol) in anhydrousdichloromethane (150 mL) under nitrogen was added triethylamine (7.7 mL,55.2 mmol). The solution was cooled to −10° C. and then a solution ofmethanesulfonyl chloride (11.51 g, 100.5 mmol) in anhydrousdichloromethane (100 mL) was added dropwise over 30 minutes. Uponcompletion, the solution was diluted to 500 mL using dichloromethane.The solution was washed twice with saturated NaHCO₃, dried over MgSO₄,filtered, and concentrated to dryness to afford the product as acolorless oil (18.9 g, 100%).

Synthesis of Phytanyl Bromide

To a suspension of magnesium bromide diethyl etherate (25.9 g, 100.3mmol) in anhydrous diethyl ether (250 mL) under nitrogen at roomtemperature was added a solution of phytanyl mesylate (18.9 g, 50.2mmol) in anhydrous diethyl ether (200 mL) dropwise over 15 minutes. Theresulting slurry was stirred for 72 hours at room temperature. Uponcompletion, the reaction mixture was cooled to 0° C. and ice cold waterwas added dropwise until all solid dissolved and bubbling stopped.Diethyl ether (300 mL) was added, and the organic and aqueous layersseparated. The aqueous layer was back-extracted with diethyl ether (200mL). The combined diethyl ether extracts were dried on MgSO₄, filtered,and concentrated. The resulting oil was purified by columnchromatography (column 10″L×2″W; eluted with 100% hexanes) to afford theproduct as a pale yellow oil (16.3 g, 90%).

Synthesis of Diphytanyl Methanol

Magnesium turnings (1.18 g, 48.5 mmol) were heated at 250° C. in an ovenfor 1 hour and then stirred at room temperature under nitrogen for 2hours. Anhydrous diethyl ether (300 mL) and a single crystal of iodinewere added, followed by a solution of phytanyl bromide (15.2 g, 42.1mmol) in anhydrous diethyl ether (30 mL). The resulting cloudy mixturewas heated to reflux overnight. The solution was cooled (0° C.) and asolution of ethyl formate (3.9 mL, 48.5 mmol) in anhydrous diethyl ether(15 mL) was added dropwise over 25 minutes. The resulting yellowsolution was again stirred overnight. The yellow solution was cooled (0°C.) and quenched using 5M HCl (15 mL), and then hexanes (100 mL) andwater (150 mL) were added. The aqueous and organic layers were separatedand the aqueous layer back-extracted twice with hexanes. The combinedorganics were washed with water, dried on MgSO₄, filtered, andconcentrated in vacuo to dryness.

The resulting pale yellow oil was dissolved in ethanol (25 mL) andtransferred to a flask containing a solution of potassium hydroxide (2.2g, 39.2 mmol) in water (5 mL). The resulting biphasic solution wasstirred at 10° C. for 2.5 hours. Ethanol was removed in vacuo andhexanes (25 mL) and 5M HCl (35 mL) were added. The organic and aqueouslayers were separated and the organic layer washed twice with water. Thecombined organics were dried over MgSO₄, filtered, and concentrated. Theresulting pale yellow oil was purified by column chromatography (column12″L×2″W; eluted with a gradient of 100% hexanes→2%→4% ethyl ether inhexanes) to afford the product as a pale yellow oil (6.4 g, 49%).

Synthesis of Phytanyl-MC3

To a solution of diphytanyl methanol (6.4 g, 10.3 mmol) and4-(dimethylamino) butyric acid hydrochloride (2.25 g, 13.4 mmol) inanhydrous dichloromethane (60 mL) under nitrogen at room temperature wasadded EDC (2.77 g, 18.0 mmol), diisopropylethylamine (5.4 mL, 31.0mmol), and 4-dimethylaminopyridine (45 mg, 0.37 mmol). After 16 hoursthe reaction mixture was diluted with dichloromethane (75 mL). Theorganic layer was washed with saturated NaHCO₃, water, and brine, andthen dried on MgSO₄, filtered, and concentrated. The resulting yellowoil was purified by column chromatography (column 10″L×2″W; eluted witha gradient of 100% hexanes→10%→50% ethyl acetate in hexanes) to affordthe product as a pale yellow oil (3.53 g, 49%) with recovery of somephytanyl methanol (2.81 g, 44%).

Example 19 Synthesis of Pan-MC4

Phytanyl-MC4 (“Pan-MC4”) (Compound 18) having the structure shown belowwas synthesized as described in Scheme 13 below.

Synthesis of Benzyl 5-Hydroxypentanoate

A solution of δ-valerolactone (10 g, 100 mmol) in 1M aqueous sodiumhydroxide (100 mL) was heated overnight with stirring at 65° C. Thesolution was concentrated in vacuo to dryness and any residual waterremoved under high vacuum at −190° C. The resulting white powder wasbroken up and suspended in acetone (40 mL). With stirring, benzylbromide (17 g, 101.4 mmol) and tetrabutylammonium bromide (0.82 g, 2.539mmol) were added. The mixture was heated at 45° C. with stirring for 72hours, cooled, and concentrated. The resulting white oily powder wasdissolved in ethyl acetate (300 mL) and washed twice each with saturatedNaHCO₃ and brine. The organic portion was dried over anhydrous MgSO4,filtered, and then concentrated. The result was a yellow oil, which waspurified by column chromatography (column 10″L×2″W; eluted with agradient of 100% hexanes→30%→50% ethyl acetate in hexanes) to afford theproduct as a pale yellow oil (3.11 g, 15%).

Synthesis of Benzyl 5-(Methanesulfonyl)Pentanoate

To a solution of benzyl 5-hydroxypentanoate (2.01 g, 9.65 mmol) inanhydrous dichloromethane (30 mL) under nitrogen at −15° C. was addedtriethylamine (2.7 mL, 19.3 mmol) followed by a solution ofmethanesulfonyl chloride (1.5 mL, 19.3 mmol) dropwise over 20 minutes.The reaction was stirred at room temperature overnight and then dilutedto 75 mL using dichloromethane. The organic layer was washed three timeswith saturated NaHCO₃ and the combined aqueous layers backextracted withdichloromethane. The combined organic phases were dried over MgSO₄,filtered, and concentrated. The resulting dark orange oil was purifiedby column chromatography (column 5″L×1″W; eluted with a gradient of 100%hexanes→10%→20%→25% diethyl ether in hexanes) to afford the product as apale yellow oil (1.39 g, 50%).

Synthesis of Benzyl 5-(Dimethylamino)Pentanoate

Benzyl 5-(methanesulfonyl)pentanoate (1.39 g, 4.85 mmol) was allowed toreact in a 5.6M solution of dimethylamine in ethanol (100 mL) for 20hours. The solution was then concentrated in vacuo to dryness. Theresulting brown oil was purified by column chromatography (column10″L×1″W; eluted with a gradient of 100% dichloromethane→2%/0.5%→4%/0.5%MeOH/NH₄OH in dichloromethane) to afford the product as a yellow oil(0.79 g, 69%).

Synthesis of 5-(Dimethylamino)Pentanoic Acid

To a solution of 5-(dimethylamino)benzyl pentanoate (0.79 g, 33.6 mmol)in anhydrous ethyl acetate (20 mL) under nitrogen at room temperaturewas added 10% palladium on carbon (250 mg). The solution was stirredvigorously under a hydrogen atmosphere. After 16 hours additionalpalladium on carbon (100 mg) was added to encourage the reaction, and at24 hours hydrogen gas was bubbled through the solution. At 40 hours thesolution was filtered through celite and concentrated in vacuo todryness to afford the product as a yellow oil (295 mg, 60.4%).

Synthesis of Phytanyl-MC4

A solution of diphytanyl methanol (0.8 g, 1.3 mmol) and4-(dimethylamino) pentanoic acid (0.24 g, 1.7 mmol) in anhydrousdichloromethane (10 mL) under nitrogen at room temperature was added EDC(0.347 g, 1.8 mmol), diisopropylethylamine (0.67 mL, 3.9 mmol), and4-dimethylaminopyridine (45 mg, 0.37 mmol). After 20 hours additional5-(dimethylamino)pentanoic acid (0.05 g, 0.34 mmol) was added toencourage the reaction. The reaction was stirred for an additional 52hours and then diluted to 50 mL using dichloromethane. The organic phasewas washed with saturated NaHCO₃, water, and brine, and the combinedaqueous layers backextracted with dichloromethane. The combined organiclayers were dried on MgSO₄, filtered, and concentrated. The resultingyellow oil was purified by column chromatography (column 10″L×1¼″ W;eluted with a gradient of 100% hexanes→10%→50% ethyl acetate in hexanes)to afford the product as a pale yellow oil (474 mg, 51%) with recoveryof some diphytanyl methanol (348 mg, 43.5%).

Example 20 Synthesis of Pan-MC5

Phytanyl-MC5 (“Pan-MC5”) (Compound 19) having the structure shown belowwas synthesized as described in Scheme 14 below.

Synthesis of Ethyl 6-(Methanesulfonyl)Hexanoate

To a solution of ethyl 6-hydroxyhexanoate (5 g, 31.2 mmol) in anhydrousdichloromethane (115 mL) under nitrogen at −10° C. was addedtriethylamine (8.7 mL, 62.5 mmol) followed by methanesulfonyl chloride(4.8 mL, 62.5 mmol) dropwise over 1 hour. The resulting solution wasstirred at room temperature for 6 hours and then diluted to 300 mL usingdichloromethane. The solution was washed with twice saturated NaHCO₃,and the aqueous layers backextracted with dichloromethane. The combinedorganics were dried over MgSO₄, filtered, and concentrated. Theresulting dark orange oil was purified by column chromatography (column5″L×2″W; eluted with a gradient of 100% hexanes→10%→20% ethyl acetate inhexanes) to afford the product as a pale yellow oil.

Synthesis of Ethyl 6-(Dimethylamino)Hexanoate

Ethyl 6-(methanesulfonyl)hexanoate was allowed to react in a 5.6Msolution of dimethylamine in ethanol (100 mL) for 17 hours. The solutionwas then concentrated in vacuo to dryness. The resulting bright orangepaste was purified by column chromatography (column 5″L×2″W; eluted witha gradient of 100% dichloromethane→1%/0.25%→2%/0.5% MeOH/NH₄OH indichloromethane) to afford the product as a yellow oil.

Synthesis of 6-(Dimethylamino)Hexanoic Acid Hydrochloride

To a solution of Ethyl 6-(dimethylamino)hexanoate (5.85 g, 31.2 mmol) indioxane (200 mL) was added 1M NaOH (200 mL). The solution was stirredvigorously at room temperature for 2 hours and then dioxane was removedin vacuo. The resulting aqueous solution was made slightly acidic usingconcentrated HCl (15 mL). At this point, dichloromethane and ether wereused in an attempt to extract the product from solution. However, allattempts failed. Instead, water was removed under high vacuum to affordthe product as an off-white solid, a mixture of approximately 35%6-(dimethylamino)hexanoic acid hydrochloride in NaCl by weight.

Synthesis of Phytanyl-MC5

To a solution of diphytanyl methanol (1.5 g, 2.4 mmol) and 35%6-(dimethylamino) hexanoic acid hydrochloride (1.79 g, 3.2 mmol) inanhydrous dichloromethane (15 mL) under nitrogen at room temperature wasadded EDC (0.65 g, 3.4 mmol), diisopropylethylamine (1.26 mL, 7.2 mmol)and 4-dimethylaminopyridine (10 mg). After 48 hours additional 35%6-(dimethylamino)hexanoic acid (1 g, 1.8 mmol), EDC (0.32 g, 1.7 mmol)and 4-dimethylaminopyridine (15 mg) were added. After an additional 72hours the reaction mixture was diluted to 75 mL using dichloromethaneand then washed with water, saturated NaHCO₃, and brine. The combinedaqueous layers were backextracted twice with dichloromethane and thecombined organic layers dried over MgSO₄, filtered, and concentrated.The resulting yellow oil was purified by column chromatography (column1¼″W×10″L; eluted with a gradient of 100% hexanes→10%→50% ethyl acetatein hexanes) to afford the product as a yellow oil (175 mg, 10%) withsome recovery of diphytanyl methanol.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many embodiments will be apparent tothose of skill in the art upon reading the above description. The scopeof the invention should, therefore, be determined not with reference tothe above description, but should instead be determined with referenceto the appended claims, along with the full scope of equivalents towhich such claims are entitled. The disclosures of all articles andreferences, including patent applications, patents, PCT publications,and Genbank Accession Nos., are incorporated herein by reference for allpurposes.

What is claimed is:
 1. A composition comprising: a plurality of nucleicacid-lipid particles, wherein each particle in the plurality ofparticles comprises: (a) a nucleic acid; (b) a cationic lipid; (c) anon-cationic lipid; and (d) a conjugated lipid that inhibits aggregationof particles, wherein at least about 95% of the particles in theplurality of particles are electron-dense.
 2. The composition of claim1, wherein the nucleic acid is an interfering RNA.
 3. The composition ofclaim 1, wherein the nucleic acid is mRNA.
 4. The composition of claim1, wherein the non-cationic lipid is a mixture of a phospholipid andcholesterol or a cholesterol derivative.
 5. The composition of claim 1,wherein the conjugated lipid that inhibits aggregation of particles is apolyethyleneglycol (PEG)-lipid conjugate.
 6. The composition of claim 5,wherein the PEG-lipid conjugate is selected from the group consisting ofa PEG-diacylglycerol (PEG-DAG) conjugate, a PEG dialkyloxypropyl(PEG-DAA) conjugate, a PEG-phospholipid conjugate, a PEG-ceramide(PEG-Cer) conjugate, and a mixture thereof.
 7. The composition of claim6, wherein the PEG-DAA conjugate is a member selected from the groupconsisting of a PEG-didecyloxypropyl (C₁₀) conjugate, aPEG-dilauryloxypropyl (C₁₂) conjugate, a PEG-dimyristyloxypropyl (C₁₄)conjugate, a PEG-dipalmityloxypropyl (C₁₆) conjugate, aPEG-distearyloxypropyl (C₁₈) conjugate, and a mixture thereof.
 8. Thecomposition of claim 1, wherein the nucleic acid is fully encapsulatedin the particles.
 9. The composition of claim 1, wherein theelectron-dense particles comprise an inverse hexagonal (H₁₁) or cubicphase structure.
 10. The composition of claim 1, wherein the cationiclipid comprises from about 10 mol % to about 50 mol % of the total lipidpresent in the particle.
 11. The composition of claim 1, wherein thecationic lipid comprises from about 20 mol % to about 50 mol % of thetotal lipid present in the particle.
 12. The composition of claim 1,wherein the cationic lipid comprises from about 20 mol % to about 40 mol% of the total lipid present in the particle.
 13. The composition ofclaim 1, wherein the non-cationic lipid comprises from about 10 mol % toabout 60 mol % of the total lipid present in the particle.
 14. Thecomposition of claim 1, wherein the non-cationic lipid comprises fromabout 20 mol % to about 55 mol % of the total lipid present in theparticle.
 15. The composition of claim 1, wherein the non-cationic lipidcomprises from about 25 mol % to about 50 mol % of the total lipidpresent in the particle.
 16. The composition of claim 1, wherein theconjugated lipid that inhibits aggregation of the particles comprisesfrom about 0.5 mol % to about 20 mol % of the total lipid present in theparticle.
 17. The composition of claim 1, wherein the conjugated lipidthat inhibits aggregation of the particles comprises from about 2 mol %to about 20 mol % of the total lipid present in the particle.
 18. Thecomposition of claim 1, wherein the conjugated lipid that inhibitsaggregation of the particles comprises from about 1.5 mol % to about 18mol % of the total lipid present in the particle.
 19. The composition ofclaim 1, wherein greater than 95% of the particles are electron-dense.20. A pharmaceutical composition comprising a composition of claim 1 anda pharmaceutically acceptable carrier.
 21. A method for introducing atherapeutic agent into a cell, the method comprising: contacting thecell with a composition of claim
 1. 22. A method for the in vivodelivery of a therapeutic agent, the method comprising: administering toa mammal a composition of claim 1.